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Identification of the Salmonella enterica serotype
Typhimurium SipA domain responsible for inducing
neutrophil recruitment across the intestinal epithelium
Daniel M. Wall,1,2William J. Nadeau,1
Michael A. Pazos,1Hai Ning Shi,1
Edouard E. Galyov3and Beth A. McCormick1,2*
1Mucosal Immunology Laboratory, Department of
Pediatric Gastroenterology and Nutrition, Massachusetts
General Hospital, Charlestown, MA 02129, USA.
2Department of Microbiology and Molecular Genetics,
Harvard Medical School, Boston, MA 02129, USA.
3Division of Microbiology, Institute for Animal Health,
Compton, Berkshire, UK.
In human intestinal disease induced by Salmonella
leukocytes (PMNs) rapidly follows attachment of the
bacteria to the epithelial apical membrane. Previously,
we have shown that the S. typhimurium effector
protein, SipA, plays a pivotal role in signalling epithe-
lial cell responses that lead to the transepithelial
migration of PMNs. Thus, the objective of this study
was to determine the functional domain of SipA that
regulates this signalling event. SipA was divided into
two fragments: the SipAb C-terminal fragment426-684
(259 AA), which binds actin, and the SipAa frag-
In both in vitro and in vivo models of S. typhimurium-
induced intestinal inflammation the SipAa fragment
tion, whereas the SipAb actin-binding domain failed to
induce PMN transmigration. Subsequent mapping of
the SipAa domain identified a 131-amino-acid region
(SipAa3294-424) responsible for modulating PMN tran-
sepithelial migration. Interestingly, neither intracellu-
lar translocation nor actin association of SipA was
necessary for its ability to induce PMN transepithelial
migration. As these results indicate SipA has at least
two separate functional domains, we speculate that
during infection S. typhimurium requires delivery of
(S. typhimurium)
polymorphonuclear of
SipA to both extracellular and intracellular spaces to
maximize pro-inflammatory responses and mecha-
nisms of bacterial invasion.
(S. typhimurium) is a facultative intracellular pathogen
that causes a variety of diseases in humans and animals,
ranging from gastroenteritis to systemic infection. In
humans, gastroenteritis symptoms include fever, nausea,
abdominal cramps and diarrhoea. The pathophysiology of
such localized enteritis is largely characterized by move-
ment of electrolytes and water, as well as to the infiltration
of large numbers of polymorphonuclear leukocytes
(PMNs), which migrate into the intestinal mucosa and
lumen from the underlying microvasculature.
The ability of S. typhimurium to cause disease in
humans is related to its acquisition of virulence genes
termed pathogenicity islands. Encoded on the 40 kb
segment of the bacterial chromosome termed Salmonella
pathogenicity island (SPI)-1 are 25 virulence genes,
which encode structural components and secreted sub-
strates of a type III protein translocation system (Hueck,
1998; Darwin and Miller, 1999). Numerous secreted viru-
lence factors, including SopE/E2, SptP and SopB/SigD,
are translocated through the host membrane where they
collectively cause actin polymerization, membrane ruffling
and Salmonella invasion (Darwin and Miller, 1999). In
addition, three SPI-1-secreted proteins, SipB, SipC and
SipD, facilitate the translocation of secreted effector pro-
teins into the cytosol of mammalian cells (Hueck et al.,
1995; Galyov et al., 1997; Collazo and Galan, 1997; Fu
and Galan, 1998).
Interestingly, one of these effector proteins, Salmonella
invasion protein A (SipA), is not only responsible for pro-
moting actin polymerization, which facilitates bacterial
entry into epithelial cells, but also plays a role in the
induction of PMN migration across the intestinal epithe-
lium (Bourdet-Sicard and Tran Van Nhieu, 1999; Zhou
et al., 1999a; Lee et al., 2000). Early reports describing a
function for SipA found that this effector forms a complex
with T-plastin and F-actin (Zhou et al., 1999b; Galkin
et al., 2002). As a result of this interaction, SipA increases
Received 14 December, 2006; revised 5 April, 2007; accepted 12
April, 2007. *For correspondence. E-mail email@example.com.
harvard.edu; Tel. (+1) 617 726 4168; Fax (+1) 617 726 4172.
Cellular Microbiology (2007) 9(9), 2299–2313doi:10.1111/j.1462-5822.2007.00960.x
First published online 18 May 2007
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd
Page 2
the actin-bundling activity of T-plastin suggesting that this
effector protein enhances bacterial invasion by stabilizing
actin filaments in S. typhimurium-induced membrane
ruffles. More recently, it was shown that SipA acts as a
molecular staple linking adjacent actin filaments together
under the cell surface resulting in the formation of projec-
tions that surround and engulf Salmonella allowing its
entry (Higashide et al., 2002). This function of Salmonella
was localized to a C-terminal fragment of the protein that
consisted of two actin-binding arms around a central
globular domain (Higashide et al., 2002; Lilic et al., 2003).
In addition to its actin-binding role, we have also shown
SipA to be both necessary and sufficient to initiate the
cellular events that lead to PMN transepithelial migration
during acute states of active intestinal inflammation (Lee
et al., 2000; Criss et al., 2001; Silva et al., 2004). Interest-
ingly, the interaction of SipA at the apical surface of intes-
tinal epithelial cells is sufficient to initiate the cellular
events that lead to such PMN transepithelial migration. To
date, the molecular mechanism underlying these cellular
events has revealed that SipA activates a novel ADP-
ribosylation factor (ARF) 6- and phospholipase D (PLD)-
dependent lipid-signalling cascade (Criss et al., 2001)
that in turn activates protein kinase C (PKC)-a (Silva
et al., 2004), which ultimately leads to the apical secretion
of the potent PMN chemoattractant, hepoxilin A3 (Mrsny
et al., 2004). Neutrophil transit across the intestinal epi-
thelium to the luminal surface is directed by this apically
released eicosanoid as a result of a chemotactic gradient
that is formed across the epithelial tight junctional
complex. Furthermore, the central role that SipA plays in
inducing epithelial responses resulting in the transepithe-
lial migration of PMN has subsequently been validated in
two distinct in vivo models of Salmonella-induced enteritis
(Zhang et al., 2002; Barthel et al., 2003).
The recognized bifunctional features of the S. typhimu-
rium SipA effector protein have prompted us to consider
whether one functional motif or distinct motifs of the SipA
molecule account for each reported function. Prior trunca-
tion analysis of SipA has shown that the C-terminal frag-
ment, termed SipAb426-684, is responsible for the binding of
SipA to the host cytoskeletal protein, actin (Zhou et al.,
1999b; Higashide et al., 2002; Lilic et al., 2003). However,
the function of the N-terminal domain of SipA, termed
SipAa2-425, has yet to be fully elucidated. To date, the only
reported function of the N-terminal fragment has been its
involvement in type III secretion with the binding of the
chaperone, InvB (between the first 158 and 264 residues)
(Bronstein et al., 2000; Lilic et al., 2006). Collectively,
these observations led us to hypothesize that the
N-terminal SipAa2-425fragment of the SipAeffector protein
harbours the functional domain for inducing the cellular
events leading to Salmonella-elicited gastroenteritis. Con-
sistent with our hypothesis, the results presented herein
reveal that the S. typhimurium N-terminal (SipAa2-425)
domain, specifically a 131-amino-acid region we have
termed SipAa3294-424, plays an essential and direct role in
activating the epithelial signalling pathways that promote
PMN transepithelial migration. Thus, we speculate that
during infection with S. typhimurium, SipA requires
delivery to both extracellular (SipAa2-425) and intracellular
(SipAb426-684) epithelial domains in order to optimally
manipulate pro-inflammatory responses and subsequent
mechanisms of bacterial invasion, both of which are fun-
damental aspects of its pathogenesis.
SipA and SipAa caused PMN transmigration whereas
SipAb did not
In our model of SipA as a bifunctional molecule we
suggest that this protein has at least two separate
domains: one for interacting with actin, and one for its role
in inflammation. It has previously been shown and we also
noted the ability of the SipAb426-684fragment to bind actin
(Fig. 1C), whereas the N-terminal fragment, SipAa2-425,
did not (Lilic et al., 2003). We reasoned therefore that the
functional motif responsible for eliciting the cellular
events, which lead to PMN transepithelial migration, might
be associated with the N-terminal SipAa2-425fragment. To
test this notion, SipA from S. typhimurium wild-type strain
SL1344 was divided into individual SipAa2-425
SipAb426-684fragments (Experimental procedures), and
each fragment was expressed and secreted by Escheri-
chia coli F-18 (Fig. 1B), a commensal strain isolated from
a healthy human (Cohen et al., 1983). This E. coli strain
has the ability to secrete proteins through its flagellar
basal body (FBB), a structure on the cell surface which
can secrete proteins in a similar fashion to a type III
secretion system (TTSS), but it cannot translocate them
to a host cell (data not shown). Western blot analysis
confirmed that proteins were being secreted (Fig. 1B) and
not released due to lysis of the bacterial cells (data not
S. typhimurium wild-type strain SL1344 SipA, and its
SipAa2-425and SipAb426-684fragments in the E. coli F-18
commensal strain so that we could directly assess the
ability of these fragments to drive PMN transepithelial
migration in the absence of other S. typhimurium viru-
lence determinants.
As shown in Fig. 2A we found that the S. typhimurium
SipAa2-425fragment, which fails to bind actin, induced a
vigorous PMN migration response resulting in nearly 30%
more PMN transepithelial migration as compared with
monolayers stimulated with the full-length SipA protein. In
contrast, the actin-binding, SipAb426-684, fragment was dra-
matically reduced (> 80%) in its capacity to induce PMN
2300D. M. Wall et al.
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Cellular Microbiology, 9, 2299–2313
Page 3
transepithelial migration as compared with the full-length
SipA molecule. These results not only indicate that the
SipAa2-425fragment is the key functional domain that
underlies the induction of epithelial responses that lead to
PMN transepithelial migration but further suggest that dis-
tinct functional motifs of the SipAmolecule account for the
promotion of PMN transcellular signals (SipAa2-425) and
the binding to actin (SipAb426-684).
As our data indicate that the SipAa2-425fragment is
involved in the activation of a signalling cascade that
results in the directed movement of PMN across the intes-
tinal epithelium, it was important to determine if this frag-
ment could also induce the secretion of IL-8. IL-8 is the
prototypical pro-inflammatory chemokine and during
active states of intestinal inflammation this PMN chemoat-
tractant is released from the basolateral surface of intes-
tinal epithelial cells where it guides PMN recruitment to
the lamina propria and subepithelium but does not recruit
PMNs across the intestinal epithelium (McCormick et al.,
1993; 1995a,b; Mrsny et al., 2004; Kucharzik et al., 2005).
As shown in Fig. 2B, exposure of the apical surface of
polarized T84 cell monolayers with either E. coli F-18
strain expressing SipA, SipAa2-425or SipAb426-684only
induced modest levels of IL-8 secretion (< 150 pg) as
compared with wild-type S. typhimurium SL1344 and the
phorbol myristate acetate (PMA)-positive control, both of
which induced large amounts of basolateral IL-8 secretion
(1600 pg and >1150 pg respectively). These data indicate
that the SipAa2-425fragment, in the absence of other
S. typhimurium virulence determinants, does not trigger a
signal transduction pathway leading to the epithelial
secretion of IL-8. This result is not unexpected given the
fact that the primary means by which epithelial cells acti-
vate IL-8 expression in response to S. typhimurium is by
the activation of epithelial toll-like receptor 5 (TLR5) via
S. typhimurium flagellin monomers (Gewirtz et al., 2001;
Zeng et al., 2003).
SipA and SipAa2-425histopathology indicated severe
Given that the results from our in vitro model of intestinal
inflammation suggest that the SipAa2-425fragment of SipA
is responsible for eliciting PMN transepithelial migration,
we next sought to verify this observation in vivo by
employing a murine model of Salmonella-induced enteri-
Fig. 1. Cloning strategy to express the SipA
fragments from the HA-tag expression vector.
A. SipA from S. typhimurium SL1344 was
divided into the N-terminal fragment
(SipAa2-425) and the C-terminal fragment
(SipAb426-684) and the respective fragments
were cloned into the pBH HA-tag expression
vector as described under Experimental
procedures. The cloned fragments were then
expressed in the commensal organism E. coli
F-18 and used in the in vitro model of
intestinal inflammation to study PMN
B. Confirmation that the cloned fragments
(full-length SipA, SipAa2-425and SipAb426-684)
could be expressed and secreted from E. coli
F-18. The secreted proteins were precipitated
and probed on a Western blot using
antibodies against HA. Shown are the
molecular weights for SipA, SipAa2-425and
SipAb426-684, which correspond to the
predicted weights.
C. Actin co-sedimentation assay showed that
SipAb (30 kDa) was active and bound actin
(43 kDa) to give a 72 kDa band on a Western
blot probed with an antibody against the
HA-tag on SipAb.
Functional domain of SipA2301
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Cellular Microbiology, 9, 2299–2313
Page 4
tis (Barthel et al., 2003). In these in vivo experiments we
used a Salmonella dublin sipA mutant strain to express
the S. typhimurium SL1344 SipAa2-425and the SipAb426-684
fragments rather than using the SL1344 background
strain as although the SipAa2-425and SipAb426-684frag-
ments could be cloned in SL1344, unlike S. dublin they
were not secreted constitutively. The ability of S. dublin to
constitutively produce proteins from the pBH plasmid was
previously observed (Cherayil et al., 2000) and it allowed
expression of the proteins of interest in animal infections
without the need for induction by IPTG.
Groups of five mice were streptomycin treated before
oral administration of S. dublin A1 (SipA–) expressing
SipA, SipAa2-425or SipAb426-684along with a positive
enteritis control (S. typhimurium SL1344) and two nega-
tive controls [S. dublin A1 (SipA–) and physiologic buffer
HBSS+]. After 72 h of infection the mice were sacrificed
and the caeca were grossly examined.As shown in Fig. 3,
it is evident that only the caeca from the mice infected with
the S. dublin A1 (SipA–) expressing either SL1344 SipA or
SipAa2-425exhibited an inflammatory phenotype remark-
ably similar to the SL1344-positive control. Such caeca
appeared inflamed as demonstrated by their shrivelled
and translucent/pale appearance and to the touch were
hard and compressed as compared with the negative
controls. In contrast, the caeca from the SipAb426-684-
infected mice displayed gross morphological features that
were consistent with the S. dublin A1 (SipA–) and HBSS+-
negative controls. For example, the SipAb426-684-infected
caeca were of normal size, brown in appearance and did
not display a rigid architecture. Thus, these results indi-
cate that the greatest amount of inflammation occurred in
mice infected with the full-length SipA and SipAa2-425-
expressing bacteria, but not in mice infected with
SipAb426-684-expressing bacteria. Next, the level of intes-
tinal inflammation was quantified histologically in a
blinded fashion by a trained pathologist (Fig. 4A). Haemo-
toxylin and eosin (HE) staining of the proximal colon
revealed that mice infected with SL1344 and S. dublin A1
(SipA–) expressing SipA or SipAa2-425exhibited inflamed
colons with clear signs of oedema, PMN infiltration in the
lamina propria, epithelial erosion and crypt elongation
(Fig. 3). In contrast, and perhaps expected from the gross
pathology results, no signs of pronounced inflammation
were observed with the S. dublin A1 strain expressing
SipAb426-684(Fig. 3). Each of the strains used in this
mouse model of enteritis all colonized the caecum and
large colon to the same extent so that the differences
noted in the severity of intestinal pathology are not due to
differences in bacterial tissue burden (data not shown).
Fluorescent staining using an antibody specific for a
PMN surface marker (reacts with a common epitope on
Ly-6G and Ly-6C) provided a more detailed picture as to
the degree of the PMN infiltration induced by SL1344,
SipA and SipAa2-425as compared with SipAb426-684
(Fig. 3). In mice infected with the positive control strain
SL1344 or S. dublin A1 expressing SipA, and SipAa2-425,
PMNs could be detected at the tips of the villi and often in
large quantities in the lumen. However, in animals
infected with S. dublin A1 expressing SipAb426-684, PMNs
were occasionally noted and the few that were present
Fig. 2. A. Induction of physiologically directed PMN transepithelial
migration (basolateral-to-apical) by apical exposure to E. coli F-18
expressing full-length SipA, SipAa2-425or SipAb426-684. E. coli F-18, a
non-pathogen isolated from the human colon, attaches to the apical
surface but is non-invasive. This strain also fails to induce PMN
transepithelial migration (McCormick et al., 1993). Negative controls
consisted of the pBH empty vector control along with a buffer
(HBSS+) control. In contrast to the negative controls, SipAa2-425
elicited >130% of the full-length SipA transmigration response,
whereas the actin-binding fragment, SipAb426-684, elicited < 10%.
Data represent the mean and SD of triplicate samples. Experiments
were performed at least three times using cells of different
passage. *P < 0.01 when the data set for a given stimulus was
compared with the amount of PMNs migrating in response to
full-length SipA.
B. Effect of the SipA fragments on the ability to induce IL-8
secretion from T84 polarized intestinal epithelial monolayers. T84
intestinal monolayers were colonized with E. coli F-18 expressing
full-length SipA, SipAa2-425and SipAb426-684and subsequently
assessed for secretion of IL-8 via ELISA assay (see Experimental
procedures). Positive control monolayers were exposed to PMA
(1 mg ml-1) and the negative control monolayers were exposed to
the buffer HBSS+. Shown here is the quantity of IL-8 secreted
(pg ml-1) into the basolateral aspect of T84 polarized monolayers.
As we failed to detect IL-8 secretion from any of the SipA
fragments, including SipA, itself, we confirmed that polarized T84
monolayers could secrete IL-8 in response to wild-type
S. typhimurium SL1344. Data represent the mean and SD of
triplicate samples. Experiments were performed at least three times
using cells of different passage.
2302D. M. Wall et al.
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Cellular Microbiology, 9, 2299–2313
Page 5
Fig. 3. Histopathology following 72 h of infection of mice with S. typhimurium SL1344, PBS and S. dublin A1 as a control, and expressing
SipA and its truncated fragments.
A. Caeca of mice showing varying levels of inflammation. Marks on ruler denoted as 1 mm increments.
B. Haemotoxylin and eosin-stained proximal colon sections (see Experimental procedures) shown at 20¥ magnification.
C. Fluorescently stained proximal colon sections (see Experimental procedures) at 20¥ magnification.
Sections were stained with DAPI and PMNs were stained using a FITC-labelled antibody specific for PMN surface markers (Ly-6G and
Ly-6C). Photographs are representative tissues for each sample (n = at least 5).
Functional domain of SipA2303
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Page 6
failed to congregate at the tips of villi. This observation is
consistent with both negative controls. As a large amount
of PMN infiltration was observed with S. dublin A1
expressing SipA and SipAa2-425but not with S. dublin A1
SipAb426-684, we quantified the level of infiltration by per-
forming an ELISA for the PMN enzyme myeloperoxidase
(MPO) (Fig. 4B). As expected, S. dublin A1 expressing
SipAb426-684caused very little PMN infiltration into the
proximal colon (161 ng per gram of tissue). Indeed, such
levels of MPO activity were consistent with those exhib-
ited by the HBSS+-infected negative control (207 ng per
gram of tissue). The greatest levels of MPO activity were
observed with SipAa2-425-expressing bacteria and these
results mirrored what was seen in the PMN in vitro assays
(Fig. 2A), as the SipAa2-425fragment (2543 ng per gram of
tissue) was more efficient at inducing PMN transmigration
than either the full-length SipAprotein (637 ng per gram of
tissue) or the SL1344 wild-type control (2097 ng per gram
of tissue). These in vivo results therefore further substan-
tiate the important role that SipA, and in particular
SipAa2-425, play in inducing epithelial cell responses that
lead to PMN transepithelial migration. In fact, the trun-
cated SipAa2-425fragment appears much more efficient at
inducing this response as compared with the full-length
SipA molecule.
Identification of the SipAa2-425fragment responsible for
inducing PMN migration
In order to map the region of SipA responsible for causing
PMN migration the SipAa2-425fragment was further
divided into threesmaller
SipAa2152-290and SipAa3294-424. Based on our earlier work
we knew that SipA monoclonal antibodies (mAb) could
block the ability of S. typhimurium SipA to drive PMN
transepithelial migration. Thus, we used this information
as an initial screening strategy employing Western blot
analysis to determine which of the SipAa2-425truncation
fragments could bind the SipA mAb. As shown in Fig. 5A
we found that the SipA mAb only bound to the truncation
region designated SipAa3294-424. The possibility that the
region responsible for induction of PMN migration is in the
SipAa3294-424fragment agrees with previous observations
that towards the N-terminal, SipAa2-425contains the func-
tional regions for chaperone binding and secretion (Bron-
stein et al., 2000; Lilic et al., 2006).
Therefore, based on this information, SipAa2-425was
divided into two fragments termed SipAa122-290and
SipAa3294-424and evaluated for their ability to induce tran-
sepithelial migration of PMN in both in vitro and in vivo
models. First, in vitro PMN transmigration assays
revealed that the fragment bound by the SipA mAb,
SipAa3294-424, and not the SipAa122-290fragment was
responsible for PMN migration (Fig. 5B). Levels of PMN
migration induced by the SipAa3294-424fragment were con-
sistent with both wild-type strains (S. typhimurium and
S. dublin), and were five times higher than those induced
by both the SipAa122-290and S. dublin SipA–strains.
Fig. 4. A. Colonic histopathology of different groups at 3 days post
bacterial infection. Disease score of colonic inflammation is shown.
The scores are assessed by determination of infiltration of
inflammatory cells (range from 0 to 4), together with the evaluation
of colon tissue damage (also a range of 0–4), with 0 scored normal
and a score of 4 showing the most significant level of disease
pathology. The data shown are measurements of individual mice
(n = 6–8 per group, 3 for PBS control) pooled from three
independent experiments.
B. PMN infiltration into the proximal colon quantified by MPO.
Tissue MPO was quantified in the proximal colon sections in mice
after a 72 h infection with S. dublin SipA–strains expressing either
full-length SipA, SipAa2-425or SipAb426-684. The positive MPO control
is shown for infection with wild-type S. typhimurium SL1344, and
the negative control represents oral gavage with only the buffer
PBS. Data are expressed as the mean and SD and represent
groups of four mice per each data set. Experiments were
performed at least three times. *P < 0.01 when the data set for a
given infection was compared with the amount of MPO in response
to the negative PBS control; NS, not significantly different from
MPO activity present in the negative control.
2304 D. M. Wall et al.
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Page 7
These observations were further tested in the in vivo
streptomycin mouse model where S. dublin strains
expressing the various fragments were used. Again,
based on results of the histopathological analysis as well
as in the quantification PMNs within the tissue (Figs 6 and
7), the SipAa3294-424-secreting S. dublin caused dramati-
cally more intestinal inflammation. HE staining of the
proximal colon sections also revealed infiltration of PMNs
into both the submucosa and the mucosa of mice infected
with the wild-type and SipAa3294-424-expressing S. dublin
A1 (SipA–) (Fig. 6). This was in contrast to the negative
controls [S. dublin A1 (SipA–) and PBS] and the S. dublin
expressing SipAa122-290. Fluorescent staining for PMN
markers confirmed the inflammation was more pro-
nounced in both the wild-type and SipAa3294-424strains
(Fig. 6) with large numbers of PMNs being seen in the
crypt. Furthermore, the quantification of tissue PMNs via
MPO analysis was consistent with these findings as the
SipAa3294-424fragment (2392 ng per gram of MPO)
induced similar levels of tissue MPO as compared
with wild-typeS. dublin (2733 ng g-1) whereasthe
SipAa122-290(438 ng g-1) results mirrored those of the
SipA mutant strain (396 ng g-1; Fig. 7).
DsipB and DsipC mutants showed SipA did not need to
be translocated to have an effect on PMN transepithelial
S. typhimurium SipA protein could trigger the PMN migra-
tion response in the absence of the type III secretion and
translocation factors, such as SipB and SipC, suggesting
that this effector may not need to be translocated into the
cytosol to initiate the events that lead to PMN transepi-
thelial migration (Lee et al., 2000). To validate this obser-
vation we took a genetic approach to determine whether
intracellular translocation or actin association of SipA is
necessary for its ability to induce PMN transepithelial
migration. Thus, two S. typhimurium non-polar deletion
DsipB(JD14) and
constructed. These strains express native SipA from the
chromosomal sipA locus and are capable of secreting
effector proteins, but cannot translocate them into the
host cell cytosol (Fig. 8A). Thus, without the SipB and
SipC translocases, SPI-1 effector proteins are merely
secreted into the extracellular milieu where they can come
into contact with the extracellular environment of the host
The secretion profile of SipA from the non-polar dele-
tion strains, DsipB (JD14) and DsipC (JD15), was quan-
tified and the results confirmed that both of these strains
secrete identical amounts of SipA compared with the
parent wild-typeS. typhimurium
(Fig. 8B). We next examined the abilities of both the
DsipB (JD14) and DsipC (JD15) S. typhimurium strains
to induce PMN transmigration. As shown in Fig. 8C, both
DsipB (JD14) and DsipC (JD15) successfully induced
PMN transepithelial migration > 80% and 70%, respec-
tively, when compared with the E. coli F-18-negative
control (< 10%). Although the response induced by the
DsipC (JD15) strain was slightly less than the response
induced by the DsipB (JD14) strain (Fig. 8C), SipB and
SipC have been shown to possess effector activities,
which may affect the ability of SipA to stimulate PMN
transmigration (McGhie et al., 2001). Furthermore, the
bacteria used in these assays were grown such that
their ability to secrete SipA and induce PMN transmigra-
tion was dependent on the SPI-1 secretion system, not
on the SPI-2 secretion system (data not shown; Bajaj
et al., 1996). Thus, these observations provide important
genetic-based evidence to further substantiate our con-
tention that SipA does not need to be translocated into
the epithelial cell cytosol to stimulate pro-inflammatory
signal transduction pathways.
previousresults demonstrated that purified
DsipC(JD15), were
strain (SL1344)
Fig. 5. A. Western blot showing binding of the monoclonal antibody
against SipA to the SipAa3294-424fragment of SipA. A GroEL loading
control is also included.
B. Induction of physiologically directed PMN transepithelial
migration (basolateral to apical) by apical exposure to
S. typhimurium SL1344, S. dublin wild type (WT) (2229), S. dublin
SipA–and S. dublin SipA–expressing SipAa, SipAa122-290or
SipAa3294-424. Controls consisted of fMLP as a positive control and
HBSS+as a negative control. S. dublin expressing SipAa3294-424had
levels of PMN transmigration that were five times higher than those
of SipAa122-290-expressing S. dublin. Data represent the mean and
SD of triplicate samples. Experiments were performed at least
three times using cells of different passage. *P < 0.01 when the
data set for a given stimulus was compared with the amount of
PMNs migrating in response to S. dublin WT; NS, not significantly
different from the response elicited by S. dublin WT.
Functional domain of SipA 2305
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Journal compilation © 2007 Blackwell Publishing Ltd, Cellular Microbiology, 9, 2299–2313
Page 8
Fig. 6. Histopathology following 72 h of infection of mice with S. dublin WT, S. dublin SipA–and HBSS+as controls, and also S. dublin SipA–
expressing SipAa122-290and SipAa3294-424.
A. Caeca of mice showing varying levels of inflammation. Marks on ruler denoted as 1 mm increments.
B. Haemotoxylin and eosin-stained proximal colon sections (see Experimental procedures) shown at 20¥ magnification.
C. Fluorescently stained proximal colon sections (see Experimental procedures) at 20¥ magnification.
Sections were stained with DAPI and PMNs were stained using a FITC-labelled antibody specific for PMN surface markers (Ly-6G and
Ly-6C). Photographs are representative tissues for each sample (n = at least 5).
2306 D. M. Wall et al.
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Journal compilation © 2007 Blackwell Publishing Ltd, Cellular Microbiology, 9, 2299–2313
Page 9
To understand how the S. typhimurium effector protein
SipA induces inflammatory responses in the intestinal
mucosa, the SipA protein was divided into (i) the
N-terminal fragment (SipAa2-425), the function of which,
apart from containing the signal sequence and binding the
chaperone, InvB, is largely unknown, and (ii) the
C-terminal (SipAb426-684) fragment, which has been shown
to bind actin and facilitate bacterial entry. Our results
demonstrate that the SipAa2-425fragment, and more spe-
cifically the SipAa3294-424region, is the key functional
domainthat underliesthe
responses that lead to PMN transepithelial migration, sug-
gesting that distinct functional motifs of the SipA molecule
account for the promotion of PMN transcellular signals
(SipAa2-425) and the binding to actin (SipAb426-684). In addi-
tion, we found that neither intracellular translocation nor
actin association of SipA was necessary for its ability to
induce PMN transepithelial migration. This observation is
consistent with the fact that secretion of type III effector
proteins in Salmonella occurs without coupled transloca-
tion (Collazo and Galan, 1997). Thus, we speculate that
during infection with S. typhimurium, SipA requires deliv-
ery to both extracellular (SipAa2-425) and intracellular
(SipAb426-684) epithelial domains in order to optimally
manipulate pro-inflammatory responses and subsequent
mechanisms of bacterial invasion.
Type III secretion systems play a central role in viru-
lence, directing the secretion and translocation of several
bacterial effector proteins into the host cytoplasm (Hueck,
1998). As SipA is a type III secreted protein it was sur-
induction ofepithelial
prising that extracellular SipA, and in particular the
SipAa2-425fragment, could induce PMN transmigration.
Although this appears to deviate from the paradigm of the
location of action of type III effectors, it should not be
over-looked that original studies investigating Salmonella
type III effectors reported that 80–90% of secreted effec-
tor proteins from Salmonella were found either associated
with non-adherent bacteria or in the infection media,
whereas only about 10% were actually translocated into
the host cell (Collazo and Galan, 1997). Consequently,
our findings raise the interesting possibility that SipA may
target two separate epithelial domains, as host cellular
translocation was not necessary for SipA to cause
inflammation. This notion is supported by evidence dem-
onstrating that although two translocation mutants of
SL1344, DsipB and DsipC, could secrete but not deliver
Fig. 7. PMN infiltration into the proximal colon quantified by MPO.
Tissue MPO was quantified in the proximal colon sections in mice
after a 72 h infection with S. dublin SipA–strains expressing either
SipAa122-290or SipAa3294-424with S. dublin WT, S. dublin SipA–and
HBSS+as controls. Data are expressed as the mean and SD and
represent groups of three mice per each data set. Experiments
were performed at least three times. *P < 0.05 when the data set
for a given infection was compared with the amount of MPO in
response to the S. dublin WT; NS, not significantly different from
MPO activity present in S. dublin WT samples.
Fig. 8. SipA activity from strains of S. typhimurium unable to
translocate effector proteins.
A. Coomassie gel showing the protein secretion profile of the DsipB
(lane 2) and DsipC mutants (lane 3).
B. Secretion of SipA from SL1344, DsipB (JD14), DsipC (JD15) and
E. coli F-18 as detected by immunoblot described in Experimental
C. PMN transmigration in response to apical infection with SL1344,
DsipB (JD14), DsipC (JD15) and E. coli F-18. Data represent the
mean and SD of triplicate samples. Experiments were performed at
least three times using cells of different passage. *P < 0.05 and
**P < 0.01 when the data set for a given stimulus was compared
with the amount of PMNs migrating in response to wild-type
S. typhimurium SL1344; NS, not significantly different from the
response elicited by S. typhimurium SL1344.
Functional domain of SipA 2307
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Page 10
SipA into the host cell, PMN transepithelial migration still
occurred. Although we have previously shown that inter-
action of the SipA protein with the epithelial cells is suffi-
cient to induce epithelial cell responses that elicit PMN
transmigration, here we have taken a genetic approach to
validate these earlier findings (Lee et al., 2000).
Therefore, on the balance of these observations we
propose a model to suggest that during infection with
S. typhimurium, SipA might exhibit a dual function requir-
ing delivery to both extracellular (SipAa2-425) and intracel-
lular spaces (SipAb426-684) based on how SipA is secreted
from the bacteria (Fig. 9). For example, in this model the
invasion ability of SipA would be mediated by the type III
protein secretion and translocation system encoded by
SPI-1, whereas the PMN transmigration inducing ability of
SipA would be independent of SPI-1. Given the observa-
tion that SipA can be secreted through the FBB of
S. typhimurium (R.A. Murray and B.A. McCormick,
unpubl. obs.) it is possible that the secretion of SipA via
the FBB system would not translocate SipA into the
cytosol, and thus would not affect actin polymerization.
This model would be consistent with the recent findings of
Higashide and Zhou (2006) showing that the SopA effec-
tor protein is not translocated into host cells following the
flagellar export of SopA. Further investigation will be
required to validate this model.
The fact that SipA exhibits bifunctional activities is
not without precedent as other type III secreted effectors
can exhibit dual functions. For example, although the
S. typhimurium translocases SipB and SipC play crucial
roles in protein translocation, these secreted proteins also
act as effector proteins once they enter the host cell. In
particular, once SipB enters the host cell it activates the
enzyme caspase-1 (Collazo and Galan, 1997) while SipC
possesses functional domains for translocation and actin
nucleation (Hayward and Koronakis, 1999; Chang et al.,
2005). Furthermore, the Pseudomonas aeruginosa effec-
tor proteins, ExoS and ExoT, are examples of bifunctional
secreted effectors with two independent enzyme activities
that exhibit two different functions interfacing with eukary-
otic cells; a Rho-Gap domain is encoded within the
N-terminus and causes actin reorganization, while the
C-terminus encodes the ADP-ribosyltransferase domain
(Sun and Barbieri, 2003).
Moreover, our results have further identified the func-
tional motif of the SipAa2-425domain responsible for elic-
iting the cellular events leading to PMN transepithelial
migration, and this has not been previously recognized.
PMN transmigration assays and infections using the
streptomycinmouse model
SipAa3294-424fragment of SipA as being responsible for
inflammation. This result is consistent with previous
observationsthat theN-terminal
SipAa122-290, contains the signal sequence for secretion
and also the region bound by the SipA chaperone protein
InvB (Bronstein et al., 2000; Lilic et al., 2006). Further-
more, the SipAa2-425fragment was also found to cause
more inflammation in the in vivo model of Salmonella-
induced enteritis when compared with the full-length SipA
protein. One explanation that may account for this obser-
vation is that the parent SipA protein (87 kDa) breaks
down to a prominent 51 kDa degradation product (Hueck
et al., 1995), which harbours the SipAa2-425domain (D.M.
Wall and B.A. McCormick, unpubl. obs.). Thus, this
smaller fragment may be in a conformation that potentially
makes the interaction of the SipAa2-425domain with the
cell surface more efficient and correlates with the
increased intestinal inflammation. It was also noted that
the efficiency of this interaction was more pronounced in
bothdetected the
and subsequent
Fig. 9. A model for the interaction of S. typhimurium with the epithelial cell monolayer.
A. S. typhimurium in the lumen secretes SPI-1 effector proteins even before it comes into contact with the epithelial cell surface. These
effectors are released through both the FBB and the SPI-1 TTSS.
B. S. typhimurium attaches to the host cell. Proteins secreted via the TTSS are translocated into the host cell cytosol while those secreted by
the FBB fail to translocate into the host cell resulting in extracellular location where secreted SipA in the lumen comes into contact with its
putative receptor.
C. Actin bundling by SipA and other translocated effectors results in the uptake of the bacterium but also PMNs begin to cross the epithelium
in response to the chemotactic gradient that is an end-product of the SipA surface-activated signalling cascade.
2308 D. M. Wall et al.
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Page 11
the mouse infection model but this is most likely do to
saturating concentrations of proteins achieved under in
vitro conditions. Thus, while the SipAa2-425fragment is
observed to be more efficient in causing inflammation in
vivo this effect is probably masked in vitro by the high
concentration of protein being secreted by the bacteria as
PMN migration proceeds at its maximum level. These
hypotheses remain to be tested.
Based on these observations, however, it is tempting to
speculate that the SipAa2-425fragment interacts with a
receptor on the apical surface of the intestinal epithelium.
Strengthening this speculation, in part, is our finding that
SipA localizes to the membrane insoluble fraction follow-
ing apical exposure to either SipA episomally expressed
in E. coli F-18 or purified SipA (W.J. Nadeau and B.A.
McCormick, unpubl. data). Indeed, other bacterial effector
proteins have been shown to affect signal transduction
pathways by acting at the cell membrane. As an example,
YpkA, a Yersinia-secreted Yop effector, localizes to the
inner surface of the eukaryotic cell membrane, where it is
thought to interfere with a signal transduction pathway
(Cornelis and Wolf-Watz, 1997). However, in contrast to
our findings with SipA, extensive studies confirm that puri-
fied Yops do not modulate biological activity if added
directly to eukaryotic cells, although they are capable of
inducing cytoxicity if artificially introduced into these cells
(Cornelis and Wolf-Watz, 1997). Comparisons between
SipA and the Shigella flexneri-secreted protein IpaA also
emphasize the unique properties of SipA. IpaA shares
approximately 20% homology with S. typhimurium SipA.
Although IpaA has also been found to affect actin poly-
merization, it appears to do so by rapidly associating with
vinculin, a protein involved in linking actin filaments to the
plasma membrane. In addition, ipaA mutants do not
exhibit the same phenotype as sipA mutants in our inflam-
matory model system as we found that ipaA mutants are
capable of inducing PMN transmigration as efficiently as
wild-type S. flexneri (A.T. Maurelli and B.A. McCormick,
unpubl. obs.). Of course, it is also possible SipAa2-425may
be able to bind and cross the epithelial cell membrane,
entering the host cell cytosol by a process that does not
require the type III translocation system. In the cytosol,
SipA may interact with one or more host cell components
to stimulate ARF-6, which ultimately leads to the produc-
tion of hepoxilin A3as well as alter the actin cytoskeleton.
In summary, this work further underscores the important
role of the effector protein SipA as a coordinator of an
immune inflammatory response resulting in PMN influx.
Byidentifying thefunctional
(SipAa3294-424) that triggers host transcellular signals
leading to the directed movement PMN across the intes-
tinal mucosa, we can start to pinpoint a potential target for
therapeutic intervention of this inflammatory disease and
perhaps of other diseases at mucosal surfaces (e.g.
domainof SipAa
Crohn's disease and ulcerative colitis) where chronic
states of intestinal inflammation are aberrantly activated.
Experimental procedures
Tissue culture
T84 intestinal epithelial cells (American Type Culture Collection,
Rockville, MD) (Passages 50–70) were grown in a mixture of
Dulbecco's modified Eagles medium and Ham's F12 Nutrient
mixture supplemented with 14 mM NaHCO3, 15 mM Hepes
buffer (pH 7.5), 40 mg l-1penicillin, 8 mg l-1ampicillin, 90 mg l-1
streptomycin and 5% fetal bovine serum. T84 cell monolayers
were grown and maintained on inverted 0.33 cm2ring-supported,
(Costar, Cambridge, MA) as previously described (Dharm-
sathaphorn and Madara, 1990) with modifications (Madara et al.,
1992). Steady-state transepithelial resistance (TER) was mea-
sured using Millipore Millicell-TERS with 'chopstick' electrodes
(World Precision Instruments, Sarasota, FL). A confluent mono-
layer corresponded to 400–1500 cells cm-2, consistent with mea-
surements by Dharmsathaphorn and Madara (1990).
filters(cell cultureinserts)
Bacterial strains, plasmids and growth conditions
Salmonella and E. coli strains used in this study are listed in
Table 1. All S. typhimurium strains are derivatives of SL1344, a
wild-type strain that is invasive in T84 cells. F-18 is a normal
human faecal isolate of E. coli, which is non-invasive and aviru-
lent but has the ability to secrete proteins through its FBB. All
bacterial strains were constructed (Murray and Lee, 2000) and
cultured in LB as previously described (Lee and Falkow, 1990).
Strains carrying pBH (Roche, Indianapolis, IN) or its derivatives
were cultured in media containing 50 mg ml-1ampicillin and
protein expression was induced where appropriate with 1 mM
isopropyl-beta-D-thiogalactopyranoside (IPTG). These growth
conditions preferentially induce the expression of the SPI-1 TTSS
and repress expression of the SPI-2 system (Bajaj et al., 1996).
Construction of DsipB and DsipC strains
Non-polar mutant deletions in sipB and sipC designed to delete
the codons for amino acids 1–351 and 1–251 of sipB and sipC,
respectively, were constructed by the PCR-ligation-PCR method
(Ali and Steinkasserer, 1995) and were cloned into the
temperature-sensitive suicide vector, pMAK705 (Hamilton et al.,
1989), generating pMAK-sipB and pMAK-sipC. Oligonucleotides
used toconstructpMAK-sipB
pMAK-sipC (5′-TTTAAGCTTGTGGCCGATGAAATTGTGAAG-
CCCAGTTCGCCATCAG-3′) were obtained from Invitrogen
(Carlsbad, CA). A 1.3 kb XbaI–KpnI fragment carrying the dele-
tion in sipB was inserted into the XbaI–KpnI-digested pMAK705
generating plasmid pMAK-sipB. A 1.3 kb HindIII–BamHI frag-
ment carrying the deletion in sipC was inserted into the BamHI–
Functional domain of SipA2309
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Page 12
plasmids were introduced into S. typhimurium strain SL1344 by
electroporation, and recipient bacteria that had integrated the
plasmids into the chromosome by homologous recombination
were selected for by their resistance to chloramphenicol at 42°C.
After passage under non-selective conditions, clones that had
resolved the co-integrate by excision of the vector sequences
were selected for by their sensitivity to chloramphenicol.
Chloramphenicol-sensitive resolvants were screened for the
correct deletion by PCR and restriction endonuclease digestion.
S. typhimurium strain JD14 contained the correct deletion in sipB.
S. typhimurium strain JD15 contained the correct deletion in
Construction of plasmids
The vector pBH (Roche, Indianapolis, IN) was used to clone and
add a haemaglutinin (HA) tag to SipA and the truncated SipA
fragments SipAa2-425, SipAb426-684, SipAa12-149, SipAa2132-290,
SipAa122-290and SipAa3294-424. Genomic DNA from S. typhimu-
rium SL1344 was used as a template in PCR reactions. Forward
primers were designed with a HindIII site and reverse primers with
an EcoRI site for cloning in frame into the multiple cloning site
(MCS) of pBH. Whole sipA was amplified using HindIIISipA2
GGACTC-3′) and SipAEcoRI
AATTCGC-3′) primers. The sipAa2-425(1–1275 bp) fragment was
amplified usingthe HindIIISipA2
sipAb426-684(1276-2052 bp) fragment was amplified using the
1275HindIII (5′-AGGGCAAAGCTTGTGAAACAACCTCTT-3′)
and SipAEcoRI primers. The sipAa12-149(1–447 bp) was am-
plified using the HindIIISipA2 and SipAa1EcoRI (5′-TCCA
(453–870 bp)
SipAa2HindIII (5′-GCGGAAAGCTTGCTGGAAAACCTGGAA-3′)
and SipAa2EcoRI primers. The sipAa122-290(4–870 bp) fragment
was amplified using the HindIIISipA2 and SipAa2EcoRI (5′-
The sipAa3294-424
(882–1272 bp) was amplified using the
SipAa3HindIII (5′-CAGAACCTACGGGTATAAGCTTCAATATTAA
TAT-3′)and SipAa3EcoRI(5′-CAAAAGAGGTTGAATTCCC
CGTAGTGCCCTT-3′) primers. Cloning of the sipA, sipAa2-425,
the wasamplified
sipAb426-684, sipAa122-290and sipAa3294-424fragments into pBH
yielded the plasmids pSipA, pSipAa, pSipAb, pSipAa12 and
pSipAa3 respectively. Clones were transformed into E. coli DH5a
to ensure expression of the truncated proteins before being trans-
formed into E. coli F-18 (Fig. 1) for use in infection studies (Sam-
by electroporation (O'Callaghan and Charbit, 1990).
Expression of SipA fragments and preparation of
extracellular proteins
Approximately 30 ml of bacteria was grown overnight according
to standard conditions. Protein production in E. coli F-18 was
induced by addition of IPTG and the culture grown with shaking
for 4 h. The bacterial culture was pelleted and the supernatant
was filtered through an Acrodisc 0.2 mm syringe filter (Pall
Gelman Laboratory, Ann Arbor, MI). Proteins were precipitated
with 15% trichloroacetic acid on ice for 1 h, collected via centrifu-
gation at 15 000 r.p.m. for 15 min at 4°C, and washed with 100%
acetone. After centrifugation for a further 5 min at 15 000 r.p.m.,
the pellet was dried for 5 min at room temperature and dissolved
in SDS sample buffer and used immediately or stored at -80°C
for later use.
SipAb-Actin co-sedimentation assay
Co-sedimentation assays were performed according to the
method of Lilic et al. (2003). Briefly, assays were performed by
mixing 20 mg of rabbit muscle G-actin (Sigma) and 50 mg of
E. coli lysate containing overexpressed HA-tagged SipAb; these
were incubated for 8 h at 4°C. Samples were centrifuged at
100 000 g for 1 h at 4°C and supernatants and pellets were
analysed by SDS-PAGE.
SDS-PAGE and Western blotting
Samples were separated through an 8–16% gradient Tris-HCl
Ready Gel (Bio-Rad, Hercules, CA) by polyacrylamide electro-
phoresis (SDS-PAGE) and transferred to nitrocellulose. Immuno-
blots were performed using a 1:5000 dilution of murine anti-HA
Table 1. Strains and plasmids used in this study.
Strains/plasmids DescriptionReference/source
S. typhimurium SL1344
S. typhimurium JD14
S. typhimurium JD15
S. dublin A1
E. coli F-18
E. coli DH5a
Wild-type hisG xyl rpsL
SL1344 DsipB
SL1344 DsipC
Wild type
endA1hsdR17(rk-mk+) glnV44 thi-1 recA1 gyrA(Nalr) relA1D(lacIZYA-argF)
HA-tag expression vector
pBH with sipA inserted in frame
pBH with sipAa2-425inserted in frame
pBH with sipAb426-684inserted in frame
pBH with sipAa12-149inserted in frame
pBH with sipAa2132-290inserted in frame
pBH with sipAa122-290inserted in frame
pBH with sipAa3294-424inserted in frame
Hoiseth and Stocker (1981)
This study
This study
This study
Cohen et al. (1983)
This study
This study
This study
This study
This study
This study
This study
2310 D. M. Wall et al.
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Journal compilation © 2007 Blackwell Publishing Ltd, Cellular Microbiology, 9, 2299–2313
Page 13
mAb (Covance, Richmond, CA). Goat anti-mouse IgG labelled
with horseradish peroxidase (Santa Cruz Biotechnology, Santa
Cruz, CA) diluted 1:2000 was used to detect the bands, which
were visualized by enhanced chemiluminescence using the
Super Signal ECL kit (Pierce, Rockford, IL). Samples were also
electrophoresed on separate gels and Coomassie was stained to
confirm equal protein loading between samples.
PMN transepithelial migration assay
The physiologically directed (basolateral to apical) PMN transepi-
thelial migration in response to bacterial stimulus has been pre-
viously described in detail (McCormick et al., 1993). Briefly,
human peripheral PMNs were isolated from anticoagulated
human whole blood by a gelatin sedimentation technique as
previously described (Parkos et al., 1991). T84 cells were stimu-
lated at the apical surface with S. typhimurium, S. dublin or E. coli
F-18 at a multiplicity of infection (moi) of 375 for 1 h. Formyl-
Methionyl-Leucyl-Phenylalanine (fMLP), a neutrophil chemoat-
tractant, was used as a positive control in all assays. Following
extensive washing, neutrophils (1 ¥ 106) were added to the baso-
lateral surface and allowed to transmigrate through the mono-
layer for 2 h. PMN transmigration was quantified by assaying for
the PMN-specific azurophilic granule marker MPO as previously
described (Parkos et al., 1991). PMN cell equivalents were esti-
mated from daily PMN standards as the number of PMN that had
completely traversed the monolayer into the apical chamber. The
use of human volunteers in this study was in accordance with
appropriate guidelines and was approved by the Massachusetts
General Hospital's Institutional Review Board for the Protection
of Human Subjects (approval #977392).
IL-8 secretion assay
Transwells coated with T84 cells in the inverted fashion were
washed with HBSS+and left for 20 min at 37°C. Transwells were
inverted in a moist chamber at 37°C and infected with bacteria at
an moi of 350. The positive control was incubated with 1 mg ml-1
PMA in HBSS+. Incubation was carried out for 1 h after which all
transwells were washed three times in warm HBSS+. Wells were
then placed in a new plate and incubated in HBSS+for 5 h. One
hundred microlitres from top reservoir (basolateral) was removed
and IL-8 secretion was measured using the Human IL-8 Elisa Kit
(Pierce Endogen, Rockford, IL).
Mice infections
Six- to eight-week-old female C57BL/6 mice were infected using a
et al.,2003).Waterandfoodwerewithdrawn4 hpriortotreatment
with 3.75 mg of streptomycin intragastrically. The following day,
water and food were again withdrawn for 4 h before the mice were
infected with 5 ¥ 107colony-forming units (cfu) of S. typhimurium
SL1344orS. dublin(500 mlofasuspensioninPBSintragastrically
by gavage). S. dublin were not induced with IPTG as S. dublin
produced the proteins constitutively. Mice were left for 72 h before
sacrifice and removal of the colon and caecum for examination.
Histological procedures
Proximal colon samples were placed in optimal cutting tempera-
ture compound (OCT) (Sakura, Torrance, CA) and snap was
frozen in liquid nitrogen. Cryosections (5 mm) were mounted on
glass slides before either HE or fluorescent staining. HE staining
was carried out according to the method of Chang et al. (2005).
Briefly, fluorescent staining was carried as follows. Slides were
fixed in acetone on ice for 10 min before submerging in a 1%
bovine serum albumin (BSA) solution. Slides were blocked by
adding first an avidin solution in 1% BSAfor 15 min, washing, and
then biotin in 1% BSA for a further 15 min (Vector Laboratories,
Burlingame, CA). Sections were stained using a fluorescein-iso-
thiocyanate (FITC)-labelled anti-mouse Ly-6G and Ly-6C (Gr-1)
antibody (BD Biosciences Pharmingen, Franklin Lakes, NJ) for
neutrophils for 90 min. After washing, DNA was stained and
mounted using the 4′ 6′-diamidino-2-phenylindol (DAPI) contain-
ing solution Vectashield (Vector Laboratories, Burlingame, CA).
Myeloperoxidase assay
Myeloperoxidase activity was measured using the Mouse MPO
Elisa test kit from Cell Sciences (Canton, MA). Briefly, sections
were taken from the proximal colon and snap was frozen in liquid
nitrogen until testing. Upon thawing sections were weighed and
then homogenizedin lysis
manufacturer. ELISA was carried out as per manufacturer's
instructions and the amount of MPO per section was expressed
as nanogram of MPO per gram of tissue.
buffer asspecified by the
Neutrophil isolation was limited to repetitive donations by five
different donors over the course of these experiments. Due to
variations in both neutrophils and transepithelial resistance
between monolayers (baseline resistance between 400 and
1500 W cm2), data were analysed within an individual experiment
and not between experiments. However, the overall trends asso-
ciated with these data are reproducible between experiments. All
results are expressed as the mean ? standard deviation of an
individual experiment performed in triplicate. P-values were cal-
culated according to Student's t-test, and values < 0.05 were
considered statistically significant.
We would like to thank Drs Bryan Hurley and Karen Mumy for
critical reading of the manuscript and James Day for construction
of strains JD14 and JD15. The research was supported by grants
from The National Institutes of Health (DK 56754 and DK 33506)
to B.A.M.
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mutagenesis: a protocol for creating gene fusions and
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Functional domain of SipA2311
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Journal compilation © 2007 Blackwell Publishing Ltd, Cellular Microbiology, 9, 2299–2313
Page 14
ment of mice with streptomycin provides a Salmonella
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Functional domain of SipA 2313
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Cellular Microbiology, 9, 2299–2313


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1. Referred pain results from the fact that cutaneous and visceral sensory pathways converge on the same spinal interneurons, so the brain cannot distinguish the original sources of pain from each other. Scotopic vision is characterized by high light sensitivity, which results from the fact that multiple rods converge on the same bipolar neurons and multiple bipolar neurons converge on the same ganglion cells, thus allowing for spatial summation in the rod pathways.
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subset: commoncrawl


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GE is the exclusive provider of a wide range of innovative products and services that are integral to staging a successful Olympic Games. GE works closely with host countries, cities and organizing committees to provide infrastructure solutions for Olympic venues including power, lighting, water treatment and transportation. GE also supplies local hospitals with diagnostic imaging equipment and healthcare technology solutions like ultrasound, MRI and electronic medical record technologies to help doctors treat athletes.
GE is an advanced technology and services company taking on the world's toughest challenges. Dedicated to innovation in energy, health, transportation and infrastructure, GE operates in more than 100 countries and employs about 300,000 people worldwide.
For more information, visit the company's Web site at www.ge.com.
Italy created both a numismatic coin programme as well as the Torino 2006 philatelic programme. For the former, 40 million €2 circulating Olympic coins were issued by La Zecca, the mint of Italy, while a series of seven stamps – six celebrating the host cities and one the mascots – comprised the latter. Nine additional stamps were then issued at the start of the Games.


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We are happy to create YouTube videos for you all your your property listings or to showcase your business. We have our own YouTube Channel that we put all of our videos on.
YouTube can be a great way to get your company name out there or to showcase a property for sale. The videos can also be embedded on your website and into a newsletter. Show off your high tech skills to your client and have a YouTube video done today.
Check out Most Wanted Advertising's 406 Productions YouTube Channel and subscribe to it for updates when new videos are posted.
Beautiful home, amazing setting 8+ acres,seasonal stream, 40x60 Shop w/office, kitchenette, bathroom, 20x40 loft, 14' garage door. The home features large kitchen w/eating area sliding doors to the deck & private back yard. Formal dining area, living room & office/den w/french doors. Master suite & private deck, mtn views, spa like master bath features custom tiled shower, jetted tub, surround sound. All of this in a park like setting. Only minutes to VA
3027 Aspenway, Helena, MT | Colette Holstine, Broker, Re/Max of Helena
3027 Aspenway, Helena, MT | Colette Holstine, Broker, Re/Max of Helena
233 sec.
Views: 2
221 McClellan Creek, Clancy, MT | Julie Millam, The Landmark Company
258 sec.
Views: 6
3225 Cabernet, Helena, MT | Christie Killeen, Helena Homes & Investments
162 sec.
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Red Roof Cafe | Alice Santos, Keller Williams Realty
102 sec.
Views: 3


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Commerce Department exposes fraudulent timeshare schemes
The Minnesota Department of Commerce has uncovered 11 unlicensed sham companies targeting timeshare owners falsely claiming to be able to sell or rent their properties and scamming them into paying thousands of dollars in transaction costs that never result in a sale or rental of their properties.
Are you a newspaper subscriber but you don't have a Digital Access account yet? https://secure.forumcomm.com/?publisher_ID=40&event=subscriber/lookup.
You will need your subscription account number and phone number. Not sure if you have an account? Email us at firstname.lastname@example.com and we can help you.


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Orioles' Hammel to Undergo Knee Surgery
(BALTIMORE) -- Baltimore Orioles pitcher Jason Hammel will have arthroscopic surgery on his right knee Monday, and is expected to miss at least a month on the field.
"Obviously, it is not what I want to do, but it is what needs to be done," Hammel said. "I can't keep pitching on a bad leg and start doing things with my arm and hurting things there."
Hammel has been having knee pain for a couple months. It finally became too much during last Friday's game versus Detroit, and he was pulled in the fourth inning.
Hammel is having a career season. The 6-foot-6 right-hander has eight wins, 106 strikeouts and 3.54 ERA in his first season with Baltimore.
Hammel has been placed on the 15-day disabled list. Pitcher Steve Johnson was called up from Norfolk to take his place.
Copyright 2012 ABC News Radio
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"The Mahdi is coming!"
Moderate Islam. Radical Islam. Militant Islam.
About the time you were getting confused and cynical about them all, along comes another term, complements of Joel C. Rosenberg: Apocalyptic Islam. … Read more
Mark's Views, Perhaps — from behind my eyeballs
"The Mahdi is coming!"
Moderate Islam. Radical Islam. Militant Islam.
About the time you were getting confused and cynical about them all, along comes another term, complements of Joel C. Rosenberg: Apocalyptic Islam. … Read more
Remorse which does not lead to repentance becomes its fatal substitute. … Read more
Are the ones in orange pictured above among these now?
"I saw the souls of them that were beheaded for the witness of Jesus, and for the word of God" (Revelation 20:4).
I don't know enough to pretend to know the eternal status of those 21 Eygptian Copts slaughtered on that beach. But I thought of the Revelation scene(s).
I also thought of this angle: … Read more
On this day, millions without natural affection celebrate: enough Supreme Court "justices" figured out a woman can do what she wants with her body.
As a result of Roe v Wade, a girl or a woman in the United States may legally…
It's her body, after all. … Read more
Night before last, as I looked for a certain old photo in one of our albums, I noticed this one:
Well, one thing has led to another for our youngest son Andy.
I don't know how he does it… … Read more
Our daughter Michayla launched her own web site.
Here are the opening paragraphs of her first blog post on December 3:
Life is made up of them. Some of them are exhilarating, others depressing. Quite a few of them are actually downright boring. There's no way of knowing when one moment will end and when the next will begin or if a moment that began in joy will end in pain, and vice versa. But one thing is true about all of them: they are fleeting.
That's part of the beauty of life. No moment lasts forever. They come and go, and though we cannot hold them, we know that more will follow, and who is to say that those will not be more lovely than the ones we cling to now? Through all the changeableness of life, through the moments we hold tightly and the ones we wish would end, there is one constant, and that is the One Who created us.
God's goodness to His people never changes. His plan of redemption never changes. And His promise of life unending will never change either. Someday, if we cling tightly to His hand and follow Him through fire and bliss alike, we will find life that has no end. We will no longer see in moments but in eternity, and all that we called beautiful in this life will be as pale glass and fallen ash in comparison.
This life is just a dressing room for that one, and moment by moment, our chances to make this life something worthwhile are slipping away.
I don't despise the moments of my life, but too often I treat them despicably.
As I commented on her blog… … Read more


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16-Year-Old David Thibault Sounds Exactly Like Elvis [Watch]
Could this be Elvis Presley reincarnated? Watch David Thibault perform 'Blue Christmas' on Quebec radio station CKOI.
Could this be Elvis Presley reincarnated? Watch David Thibault perform 'Blue Christmas' on Quebec radio station CKOI.
When "Breaking Bad" stars Bryan Cranston and Aaron Paul first read their scripts for the show's finale, they were together...AND they were being filmed.
A show called "Guinness World Records Unleashed" premieres on TruTV tomorrow night. And a woman named Trisha Paytas...who works as a LINGERIE model...will try to break the record for fastest TALKER.
People have WAY too much time on their hands. Someone actually took the time to splice together clips of David Hasselhoof so it sounds like he's singing the theme to 'The Fresh Prince of Bel-Air'.
The opening of this Sunday's episode of "The Simpsons" parodies "The Hobbit". And you can check it out a few days early on YouTube. Marge plays Gandalf, Moe plays Gollum, and Homer plays the dwarf, Gimli.
A woman named Sharidan Sotelo was an assistant editor on "Breaking Bad". And now she's reimagined the series, using professional ICE SKATERS to play all the parts.
Oh, October...such a great/scary month it is! Everybody knows that October is just a scary month in general just because of Halloween!
Ellen DeGeneres gave $10,000 to a New Hampshire waitress who paid a tab for two National Guard soldiers.
One of the best things about "South Park" is how unbelievably timely they can be . . . because they regularly write and produce new episodes in LESS THAN A WEEK, and don't turn them in until the day they air.
Superman turned 75 this summer and to mark the occasion, Warner Brothers made an animated video tracing the evolution of the character, from his first appearance in the comics in June of 1938 to the present.
You may be annoyed with some of the things your friends are posting on Facebook and Twitter about the 'Breaking Bad' finale. We are too, and thus decided to offer up a list of some of the most irritating reactions people are having towards the finale of (un)arguably the greatest show of all time.
There's a video on YouTube of a car driving up to a shirtless guy on the street who's SUPPOSEDLY a homeless guy with the ability to do amazing impressions. But it immediately seems suspicious when he starts doing characters from "Breaking Bad", which has its series finale this Sunday. Because obviously not many homeless people have access to "Breaking Bad" episodes.


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we came into this hotel recommended from a friend and the truth that we loved the spacious rooms all with sea views, the bathroom is large with respect to cleanliness is great, is beachfront and the situation is great because you have all kinds of bars and restaurants to eat and go out at night, the attention of the staff very friendly and attentive to what you need, in order to charm us.
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- Melia Costa Del Sol Torremolinos, Spain


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Character actor Than Wyenn has died, aged 95.
Which Hollywood megastar has been happily married for the last 36 years to her brother's friend?
Veteran TV screenwriter/producer Wilton Schiller has died, aged 95.
Veteran actress Juanita Moore has died, aged 99.
TV rookies 'Brooklyn Nine-Nine,' 'Sleepy Hollow' and 'Elementary' have a new spin on things.
We count down 5 of the Oscar winner's less than memorable performances.
Joe Conley, a film and television character actor known best as Ike Godsey on 'The Waltons,' passed away at 85.
What did the actress every do to you? We find out.
The comic book hero continues to be modernized — here's the proof.
Wait...Tom Hanks has a son who's a rapper?
|Credited as
|Rod La Rocque


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Presented by Home Building Association of RichmondNavigation
Click here to see a video from Richmond Homearama 2014! We look forward to seeing you in RounTrey for Richmond Homearama 2015!
Richmond Homearama 2015 will open May 1–17, 2015 from 11:00AM to 6:00PM! We can't wait to see you then!
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Chief Minister Siddaramaiah has announced a five-point programmes for the effective implementation of Article 371(J) of the Constitution, which provides special status to Hyderabad Karnataka region comprising of Gulbarga, Raichur, Yadgir, Bidar, Bellary and Koppal in the Assembly on Wednesday.
Making an impromptu statement in this regard, Mr. Siddaramaiah said a Cabinet sub-committee had been formed under the chairmanship of the Minister for Rural Development and Panchayat Raj to formulate needed guidelines and notifications for the effective implementation of the special status.
The sub-committee submitted a report along with draft rules and notifications and the State Cabinet had adopted it with some modifications. He said steps would be taken to implement the recommendations.
The government was waiting for an order to be issued by President Pranab Mukherjee to fix special responsibility on Governor H.R. Bhardwaj. Moreover Mr. Bhardwaj had to issue another notification for setting up of the Hyderabad-Karnataka Area Development Board (HKADB).
The board will advise the Governor on formulation of norms and channelising of funds for the development of the area. It would also advise the Governor on creation of posts and permission to start educational institutions.
Mr. Siddaramaiah said organisational set-ups would be created at the State, region, district and taluk-levels for effective implementation of the programmes. Effective technology would be employed for keeping a watch on the flow of funds released by the Centre, he added.


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The weapon was spotted on an X-ray scanner as the Apollo 13 star traveled through Heathrow airport to catch a Los Angeles-bound flight.
The star was briefly detained by officials for questioning but released without charge.
A police source tells Britain's Daily Express newspaper, "We can't reveal what the actor told us, but many people carry sharp objects with them for personal hygiene reasons without understanding the security implications."
COPYRIGHT 2007 WORLD ENTERTAINMENT NEWS NETWORK LTD. All Global Rights Reserved.


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"Oooh, what kind of surprise is it? Is it something I would like? Why don't you tell me about it?" Napa… will I ever get sick and tired of this place? Probably, but for now I'll enjoy every excursion until my mind grows weary of the crimson grape vines, luscious backdrops of Mt. St. Helena, … Continue reading
"Extra green sauce, ok, the total is $17.35, it will be ready when you arrive." Seriously? Another burrito joint?* I could've sworn this place was swarming with more Southern places than I can count (not that it's a bad thing, *ahem* Brown Sugar Kitchen). Granted, this was my first time glancing at the corner burrito … Continue reading
"Can you send me the menu?… **after reading it online** Come on son, I don't know about this place, you know I'm not really into this sort of thing…but it's all good." ***Author's Note*** I know the ending to this two-part series was long overdue so be prepared with my content binge in entertainment, food … Continue reading
"Welcome back brother…we haven't kicked it in a cool minute. Let me tell you what really happened…" NYC, I was long overdue to revisit this place. Not only for the establishments I failed to experience the first time around but also uncover the more intimate side of my wanderlust desires and perspective. Taking 15 hours … Continue reading


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Zoltán Gera told UEFA.com that Hungary "have a good chance" of reaching UEFA EURO 2016 while Kostas Stafylidis says Greece "are not dead yet – there's always hope".
While Faroe Islands coach Lars Olsen took plenty of positives from a 1-0 loss in Romania, match winner Claudiu Keșerü was quick to defend his side's safety-first approach.
Hungary 0-0 Greece
There were no goals in Budapest as the hosts fell further behind Group F's leading pair and Sergio Markarián's side earned their second point.
Lacazette and Payet prosper
Hamšík clinches award
Trending now
Goal of the WeekFriday 20 March 2015 8.00CET


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Amazing how the song "Tik Tok" has landed Ke$ha, a new pop sensation, worldwide recognition. And it's even more amazing to find out that watch giant, Casio, also drawn to the starlet's power. The watchmaker held an all-day event at the Manhattan Center to introduce its brand ambassadors which include Ke$ha who will represent Baby-G's women's watches. Stylistically, Casio is aligning Baby-G with the revival of Eighties fashion and Ke$ha suited the role with her edgy party-girl image.
Ke$ha has also signed to design two watches, which will appear in the advertising campaign launching for fall and the first which will be launched next summer.
Among Casio's newest ambassadors, there's also Khloe Kardashian's husband, Lamar Odom, who will represent Ultimate Tough GX56, which features the largest case design in G-Shock history. The solar-powered watch also feature a new triple-density and triple-layer structure to increase its shock resistance. Odom also hinted that he and wife, Khloe, will release a unisex perfume next year.
Here are our picks of Casio's Baby-G and G-Shock for you:
Baby-G Lady G Figure Set, $150
Baby-G Square Vivid Combi Watch, $110
Baby-G Gradiation Watch in Yellow, $122


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Blogging TV, one show at a time
Posts filtered on Tag
Tune In | 07.09.13
More Colin Firth, less Troy Barnes, and Crazy Aunt Paula joins the SYTYCD judges, FINALLY.[Read More]
'America's Got Talent': Texas singers and rain makers
Lufkin singer Dave Finley brings the rasp to AGT. [Read More]
Tune In | 07.02.13
Craig Robinson might get his own NBC sitcom. (But knowing NBC, probably not.)[Read More]
'America's Got Talent': Pants off dance off
Mini Magic Mikes and other strip teases[Read More]
Tune In | 06.25.13
So You Think You Can Dance gets on with the competition.[Read More]
UPDATED WITH VIDEO: 'America's Got Talent': Blowing us away in the Windy City
It's a specific kind of talent to balance bowls on your head while riding a 12 foot tall unicycle …[Read More]
Tune In | 06.18.13
When does Sawyer come back to TV? Not as soon as you might like, unfortunately.[Read More]
UPDATED WITH VIDEO: 'America's Got Talent': A modest proposal
The auditions come to Texas. Yee-haw.[Read More]
Tune In | 06.11.13
Hey ninjas, guess what?[Read More]
Show More


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Beautiful pitcher, It was a bit heavy for me, I gave to my granddaughter and she loves it. Like I said, nice great quality but just to heavy for me.
This pitcher goes beautifully with the blue glasses I bought earlier. It is also nice and light so you can easily lift it when it is full. I love it!
I read the other reviews to help in my decision. This looks great online & even better when you get it. I was looking for a large volume pitcher instead of the little acrylic ones. This is magnificent!! I love blue and this is so nice. After the pitcher came, I was so pleased, I bought the matching glasses. They were good too. What a bargain for the workmanship & quality. I would buy anything this company makes.
Simply Gorgeous! Has a nice weight so it seems pretty durable. Hope to enjoy this artistic pitcher for years to come.
I purchased this pitcher and matching glasses to give as wedding and shower gifts to a young bride in our family. They are so beautiful, it's going to be hard for me to give them away because I want to keep them for myself! I'm sure they will be well received because they are very lovely and probably something a newly wed couple wouldn't splurge on for themselves. The glass is very bright and clear and heavy, and feels good to the touch. They will be fun to use, and I'm sure will receive lots of compliments because of how unique they are in appearance. The spiral glass is very pretty with a true cobalt blue. Yes, there are a few bubbles, but they do not detract from the overall beauty of the glass. They were packed really well, but they were slow in arriving. I waited longer than expected, but they were worth the wait.
This matches my glasses that I purchased in Mexico while on a cruise. The set came with a pitcher, which matched exactly, and just as we landed at our home airport, my husband dropped the bag, the only broken piece was the pitcher. This one is a good substitute.
Very nice, person who received this as a gift loved it
The pitcher is quite attractive and makes a great addition to a set of matching glasses. Its only down side is its weight. It's made of sturdy glass that gives it a bit of heft when liquid is added.
This is beautiful piece and would compliment any kitchen setting.
I gave this as a house warming gift and the couple loved it. They said it was different and they enjoyed the colors and the feel of the pitcher.
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Outstanding performance of staff! Superbly designed hotel! Luxurious pool and spa. Hotel is well suited for the "spa/ wellness- traveler". - Anonymous
This hotel has an excellent location in Sanur. There are direct seaside
with long sandy beach an the same time 2 great pool with sweet water.
The breakfast was amazing: dozen of different meats, exotic fruits, jams
from the continental , English till the Indonesian breakfast. There are
2 comlimentary drinking water daily in the room as in other good hotels
of Bali. If you wish - for the dinner - you can walk easy out of the
hotel in 50-100 m you find friendly restaurants with live music. - Anonymous
To-die-for modern accommodations - gorgeous villas enclosed in
individual privacy walls, your own personal pool, cabana, lounge area
(covered and uncovered both, several lounge chairs, bar/kitchen area
with refrigerator, table, etc.) Honeymooners would never have to emerge
from their villa. Extremely friendly and attentive staff. - Anonymous
We stayed at the 3 rooms villa for 2 nights and it was fantastic. The villa is spacious, clean and well maintained although it has been there for quite some time. There were one housekeeper, a technician for swimming pool and gardener to do housekeeping the next morning to take care of the place. The structure of the villa are thoughtful with huge dressing table and outdoor and indoor shower. Comfortable bed and quiet environment and we had really good sleep. Perfect for couple/family/friends. - Anonymous
Smiling staff and very accommodating. Rooms are very nice using earthy colours and not too much wood. The outdoor shower is nice, private, yet not intrusive. I like the quietness, away from the usual noisy life. Dining prices are resonance, but drinks is about on the high side. I like ala carte style Bfast, as it's prepared a- la minute. The quality of items used adds to the experience, the soft music around the compound, at the pool. Using of Crab tree and Evelyn products, live traditional mus - Anonymous


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Retail price: $6.99
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Clear Eye Herb Tea is made of wild dendrobium (dendrobii caulis). solomon seal (polygonati rhizoma) and abalone shell (haliotidis concha). They provide dendroxine, dendramine, amino acids, calcium carbonate, etc.
Traditional Chinese medicine uses them to nurture the eye and pacify the nervous system, protect retina, and improve eyesight.
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Lipstick Lacks Gloss
September 28, 2008
By jon wise
VIRGIN'S new girlie drama Lipstick Jungle doesn't make up for the loss of Sex And The City from our screens.
Candace Bushnell's follow-up to SATC lacks the punch of its predecessor. Against rivals such as Desperate Housewives and Ugly Betty it is second rate. Brooke Shields (right) stars as film studio boss Wendy, alongside Kim Raver as magazine editor Nico, and Lindsay Price as fashion designer Victory.
While the outfits and heels are sexy and stylish, the show as a whole is watchable but missable.
(c) 2008 People, The; London. Provided by ProQuest LLC. All rights Reserved.
Topics: Kim Raver, Candace Bushnell, Lipstick Jungle, Lindsay Price, Sex and the City, Lipstick, ProQuest, Brooke Shields, Nico, Entertainment Culture


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re•duc•ti•o ad ab•sur•dum
Pronunciation: (ri-duk'tē-ō" ad' ab-sûr'dum, -zûr'-, -shē-ō"), Strict Standards: Non-static method FenSites::linkTo() should not be called statically in /site/html/dictionary/reductio-ad-absurdum.html on line 73 [key] Logic.
a reduction to an absurdity; the refutation of a proposition by demonstrating the inevitably absurd conclusion to which it would logically lead.
Random House Unabridged Dictionary, Copyright © 1997, by Random House, Inc., on Infoplease.
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Ahmed Ben Bella, Algeria's first president and one of the leading figures in its independence struggle against France, has died at the age of 95.
Ben Bella, whose death comes just weeks after the 50th anniversary of Algerian independence, passed away at his family home in Algiers on Wednesday, Algerian state media reported.
No cause of death was reported, but Ben Bella had twice been treated for respiratory problems at the military hospital of Ain Naadja.
"Today we lost one of modern Algeria's bravest leaders," said Algerian President Abdelaziz Bouteflika in a message of condolence read on state television as he announced eight days of national mourning.
Ben Bella will lie in state at Algiers' People's Palace from noon on Thursday, with his funeral due to take place on Friday, state media said.
A former soldier in the French army, Ben Bella was a key figure in Algeria's years-long revolt against French rule, serving time in prison until his release as the north African nation gained independence in 1962, and becoming president in 1963.
But he was overthrown in a military coup in 1965 and kept under house arrest until 1980. After a subsequent decade in exile in Switzerland, he was pardoned in 1990 and returned to Algeria.
Soldier and footballer
Ben Bella was born in 1916 to a peasant family in Marnia, on Algeria's border with Morocco. He joined the French army in his late teens, rising to the rank of senior warrant officer, and playing top-flight football for Olympique de Marseille.
He fought with distinction with the Free French Forces in Italy during World War II and won five French decorations including the prestigious Military Medal.
But Ben Bella turned against French rule after the war and was elected municipal councillor for the anti-colonialist "Movement for the Triumph of Democratic Liberties".
When the movement was declared illegal, Ben Bella went underground. Arrested in May 1951, he was interned but staged a dramatic escape two years later.
He fled to Cairo where he was among the architects of the 1954 uprising which began Algeria's war of independence.
Despite being imprisoned by the French, he was the acknowledged head of the independence movement throughout the revolution and became an icon for the global anti-colonial movement.
Elected president of the newly-independent nation, he ruled extravagantly and erratically for less than three years before being toppled by Colonel Houari Boumedienne, the head of the army.
Until Boumedienne's death 13 years later, Ben Bella became a "non person" in Algeria, as no public mention of his name was allowed in Algeria's state-controlled media.
Boumedienne's successor Chadli Bendje freed Ben Bella from more than a decade of detention without trial, ultimately allowing him to go abroad with his wife Zora and their two adopted daughters.
He headed the opposition Movement for the Democracy in Algeria Party, which competed in the aborted 1991 elections, winning just two per cent of the vote.
His party was banned in 1997, but he continued to live in Algeria, often condemning government policies.
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Israeli startup shatters the way we look at - and through - glass
Controlling the transparency of windows with a touch is no longer science fiction: Meet smart glass and Gauzy, the startup behind it.
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Every so often a new company changes our concept of how we utilize everyday products. Gauzy, a startup in south Tel Aviv, is revolutionizing not only the way we look at glass - but also the way we look through it.
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Ironfest #18
Ironfest 2012 Lithgow NSW Australia.
awesome shot
Thankyou very much.
– garts
Fantastic capture!
Thankyou Carol.
– garts
Great work Adrian x
Thankyou Irene.
– garts
A wonderful capture dear Adrian, and a great use of textures.x
Thankyou Catherine.
– garts
You really captured this moment! Great work!
Thankyou very much.
– garts
There's just something about knights. Cool image.
Thankyou Sybille.
– garts