MINIREVIEW

Multiple invasion mechanisms and different intracellular

Behaviors: a new vision of Salmonella–host cell interaction
s Wiedemann1,2
Zineb Boumart1,2,3, Philippe Velge1,2 & Agne
1
Institut National de la Recherche Agronomique, UMR1282 Infectiologie et Sante  Publique, Nouzilly, France; 2Universit
e Francßois Rabelais,
UMR1282 Infectiologie et Sante Publique, Tours, France; and Agence Nationale de S
3
ecurit
e Sanitaire de l’alimentation, de l’environnement et du
travail, Laboratoire de Ploufragan-Plouzane, Unite Hygiene et Qualit
e des Produits Avicoles et Porcins, Plouragan, France

Correspondence: Agnes Wiedemann, INRA Abstract

Centre Val de Loire, UMR 1282 ISP, bat 311,
Nouzilly 37380, France. Tel.: +33 2 47 42 78
70; fax: +33 2 47 42 77 79; e-mail: Agnes.
Wiedemann@tours.inra.fr
Received 30 July 2014; revised 16 September
2014; accepted 27 September 2014. Final
version published online 21 October 2014.
DOI: 10.1111/1574-6968.12614
Editor: Andre Klier
Keywords
Salmonella; invasion; proliferation.
MICROBIOLOGY LETTERS
Salmonella is a facultative intracellular bacterium found within a variety of
phagocytic and nonphagocytic cells in vitro and in vivo. For decades, it has
been accepted that Salmonella can enter cells only through a Trigger mecha-
nism mediated by a type three secretion system, called T3SS-1. However, recent
researches have shown that this bacterium can use other invasion pathways
mediating either Trigger or Zipper entry processes. Following eukaryotic cell
invasion, Salmonella has to ensure its survival and proliferation within host
cells. To do so, this bacterium resides either within a membrane-bound vacuole
or freely within host cell cytosol. It is not clear why Salmonella has developed
these alternate mechanisms for cell invasion and proliferation, but this provides
a new insight into the mechanisms leading to Salmonella-induced diseases.
Thus, the aim of this review is to show the evolution of Salmonella–host cell
interaction paradigms by summarizing the different strategies used by Salmo-
nella serotypes to invade and proliferate into eukaryotic cells.

mediated by an outer membrane protein invasin called Rck.

Introduction
Salmonella is an enteric bacterium recognized as an impor-
tant economic and public health problem throughout the
world (Hoelzer et al., 2011). Depending on the serovar and
the host, Salmonella can lead to diseases ranging from gastro-
enteritis to a life-threatening systemic infection. An essential
feature of Salmonella pathogenicity is the entry within phag-
ocytic and nonphagocytic host cells. Thus, the entry and
intracellular survival of Salmonella are considered as critical
steps for proliferation, dissemination, and transmission to
other hosts (Richter-Dahlfors et al., 1997; Salcedo et al.,
2001; Meyerholz et al., 2002; Geddes et al., 2007). In recent
years, our vision of Salmonella–host cell interaction has con-
siderably evolved. It has always been assumed that Salmonella
enters host cell via the so-called Trigger mechanism, which
requires a type three secretion system, called T3SS-1 and that
was the center of attention for over decade (Marlovits &
Stebbins, 2010). However, in vitro studies have shown that
Salmonella can use multiple ways to invade nonphagocytic
host cell (Rosselin et al., 2010). The characterization of one
of the T3SS-1-independent invasion pathway has shown the
ability of Salmonella to invade cells via a Zipper mechanism
Therefore, Salmonella is the first bacterium shown to be able
to invade host cells via both Zipper and Trigger mechanisms
(Rosselin et al., 2010). Following invasion into eukaryotic
cells, most of bacteria remain in a modified phagosome
called the Salmonella-containing vacuole (SCV). Although
the SCV has been considered the primary niche for intracel-
lular Salmonella, this bacterium seems to have a distinct
bimodal lifestyle replicating in SCV as well as in the cytosol
of epithelial cells (Steele-Mortimer, 2008; Malik-Kale et al.,
2012) (Figure 1). Nevertheless, little is known about the mul-
tiple mechanisms of entry and proliferation into host cells
and their roles in Salmonella disease and host specificity.
Thus, this review focuses on how our understanding of Sal-
monella pathogenicity has evolved over time, showing how
this bacterium manipulates the host cell using different strat-
egies for invasion and establishment of an intracellular niche.
I- Diversity of Salmonella entry
strategies: Zipper vs. Trigger
Intracellular bacteria have evolved two different strategies
to invade nonphagocytic cells by modulating the actin

FEMS Microbiol Lett 361 (2014) 1–7 ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
2 Z. Boumart et al.
cytoskeleton: the Zipper and the Trigger mechanisms.
Bacteria, such as Shigella flexneri, enter host cells via a
Trigger mechanism using a T3SS and bacterial effectors
proteins (Schroeder & Hilbi, 2008). Other bacteria such
as Listeria monocytogenes and Yersinia pseudotuberculosis
use the Zipper mechanism to invade nonphagocytic host
cells. These bacteria express a surface protein able to bind
eukaryotic surface receptors. This interaction leads to the
activation of host cell signaling pathways inducing actin
polymerization and bacterium uptake (Cossart & Sanso-
netti, 2004). However, contrary to Salmonella, none of
these bacteria has been shown to be able to invade host
cells using both Zipper and Trigger mechanisms.
The study of Salmonella host cell invasion mechanism
has been initiated by Takeuchi in 1967 (Takeuchi,
1967). Until recently, it has been assumed that Salmo-
nella invades host cells only via a Trigger entry mecha-
nism, which requires the T3SS-1, encoded by the
Salmonella pathogenicity island (SPI-1). The T3SS is a
needle complex found in many Gram-negative patho-
gens, and a high homology has been found between the
T3SS apparatus of Salmonella and Shigella (Groisman &
Ochman, 1993; Galan & Wolf-Watz, 2006). For many
years, most researches have focused on understanding
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
and describing this sophisticated invasion system, both
at the bacterial and molecular levels (McGhie et al.,
2009; Moest & Meresse, 2013). However, recent evi-
dences have demonstrated that entry strategies of Salmo-
nella serovars proved to be more diversified. Contrary
to what it has been thought, the T3SS-1 is not the sole
entry strategy of Salmonella (Coombes et al., 2005; De-
sin et al., 2009; Rosselin et al., 2010). In fact, several
studies have shown that Salmonella strains lacking SPI-1
are able to invade in vitro numerous cells and induce in
vivo intestinal pathologies both in humans and different
animal models (Hu et al., 2008; Aiastui et al., 2010).
For example, in vitro studies have shown that Salmo-
nella did not require its T3SS-1 to enter a 3-dimen-
sional intestinal epithelium (Radtke et al., 2010).
Murray et al. have also shown that in a murine model,
S. Typhimurium DSPI-1 mutant was not impaired in
the spleen infection of infected mice, suggesting that
T3SS-1 from S. Typhimurium is not involved in the
passage through the intestinal barrier and the infection
of systemic organs (Murray & Lee, 2000). Furthermore,
it has been observed that S. Typhimurium with a non-
functional T3SS-1 still has the ability to infect mice
after intraperitoneal inoculation but also chicks after
Fig. 1. Different strategies used by Salmonella
for invasion and proliferation into eukaryotic
cells. Salmonella is able to invade host cell via
both a Trigger and a Zipper mechanism. The
Trigger entry process is mediated by the T3SS-
1 effector proteins and is morphologically
characterized by important cytoskeletal
rearrangements. Following the entry
mechanism dependent of the T3SS-1,
Salmonella resides and multiplies in the SCV
that undergoes different stages of maturation,
which is partly due to the T3SS-1 effectors.
Salmonella replication is accompanied by the
formation of SIFs, that extends from the
surface of the SCV, facilitating the delivery of
nutrients to the SCV. In the case of the SCV
damage, bacteria could be destroyed
following either the SCV–lysosome fusion or
the autophagy response. Finally, a portion of
bacteria is able to escape from the SCV and
multiply efficiently in the cytosol of epithelial
cells. The Zipper mechanism mediated by Rck
induces a minor cytoskeletal actin
rearrangement, leading to the internalization
of the bacteria into a vacuole, whose
biogenesis under these conditions has not
been well studied. The PagN-mediated entry
process requires actin polymerization but it
remains poorly characterized.
FEMS Microbiol Lett 361 (2014) 1–7
Salmonella-host cell interaction
oral inoculation (Galan & Curtiss, 1989; Morgan et al.,
2004). Overall, these data suggest that other invasion
pathways are involved in Salmonella infection. Among
these mechanisms, a recent research has shown that Sal-
monella can also invade nonphagocytic cells by a Zipper
mechanism through the Rck invasin, a protein encoded
by the rck gene located on the large virulence plasmid
(Rosselin et al., 2010). This protein is not expressed in
all Salmonella serovars. It is highly conserved in nonhost
specific serovars such as S. Enteritidis and S. Typhimu-
rium, it could be found in S. Dublin, but has never
been detected in the other Salmonella serovars (Rychlik
et al., 2006; Futagawa-Saito et al., 2010).
In Salmonella, bacterial internalization mediated by
Trigger or Zipper mechanisms involves actin remodeling
likely through stimulation of host cell signaling pathways.
The Trigger invasion mechanism is mediated by the
injection of a cocktail of effector proteins into host cells
inducing directly or indirectly significant cytoskeletal rear-
rangements known as ‘membrane ruffles’ (Schlumberger
& Hardt, 2006). These events are mediated by a subset of
T3SS-1 effectors (SipA, SipC, SopB, SopE, SopE2). The
SipA and SipC proteins directly bind actin preventing its
depolymerization (McGhie et al., 2001). The SopB, SopE,
and SopE2 proteins indirectly modulate actin activity by
stimulating Rho GTPases family (Rac1 and Cdc42)
required for the initiation of cytoskeletal actin polymeri-
zation via the Arp2/3 complex, an eukaryotic factor
involved in the actin networks control (Zhou & Galan,
2001; Patel & Galan, 2006). Once inside the host cell,
another effector protein of the T3SS-1-termed SptP shows
an activity counterbalancing those of SopE and SopE2.
This effector inactivates Rho GTPases allowing the resto-
ration of actin and the return of host cell to the normal
state (Murli et al., 2001). The other T3SS-1 effectors con-
tribute to a variety of postinternalization processes such
as host cell survival and modulation of the inflammatory
response.
Unlike the Trigger entry mechanism, the Zipper entry
mechanism is mediated by an interaction of a bacterial
ligand with a host cell receptor, which induces a signaling
cascade and a local accumulation of actin, leading only to
minor cytoskeletal actin rearrangements and tight mem-
brane extensions (Cossart, 2004; Rosselin et al., 2010).
Until now, it is not clear why such a difference in mem-
brane rearrangement and actin polymerization is observed
between these two mechanisms.
In the case of Rck, its cell receptor has not been identi-
fied yet, but as observed for the Trigger mechanism, the
Rck-mediated entry involved similar host cell partners
like the small Rho GTPases Rac1 and Cdc42, which lead
to the activation of the Arp2/3 complex and the mobiliza-
tion of actin rearrangements, driving to bacterium uptake
FEMS Microbiol Lett 361 (2014) 1–7
3
(Unsworth et al., 2004; Rosselin et al., 2010; Mijouin
et al., 2012). However, contrary to the Trigger mecha-
nism, the activation of the Rho GTPases partners seems
specific to the Rck-mediated entry process. This process
is especially dependent upon the activation of tyrosine
kinase and class I PI3 kinase that activates Akt (Mijouin
et al., 2012; Wiedemann et al., 2012).
The role of Rck in Salmonella invasion has clearly been
demonstrated and found to be dependent of the cell lines
and cell types (Rosselin et al., 2011). Furthermore, the
Rck operon seems to be controlled by the SdiA protein, a
transcriptional regulator of the quorum sensing (Ahmer
et al., 1998; Abed et al., 2014). However, its contribution
in Salmonella pathogenesis remains unclear as the
mechanism inducing Rck expression in vivo is not well
understood.
In addition to Rck and T3SS-1, PagN is an another
identified invasin widely conserved in the Salmonella
genus (Lambert & Smith, 2008). It has been shown that
the binding of PagN to extracellular heparin sulfate prote-
oglycans induces invasion of Salmonella. This outer mem-
brane protein (OMP)-mediated entry process requires
actin polymerization, but it remains poorly characterized
(Lambert & Smith, 2008). HlyE a pore forming hemolysin
encoded by SPI-18 was also identified as new virulence
determinant that seems to enhance Salmonella invasion of
some cell types, but the precise mechanism of invasion
remains unknown (Fuentes et al., 2008).
In Salmonella, other unknown and nonidentified inva-
sion factors seem to be involved during cell entry. Indeed,
it has been demonstrated that a Salmonella strain not
expressing Rck, PagN, and T3SS-1 still has the ability to
enter different cell types (Rosselin et al., 2011). Micro-
scopic observations within different cell types have also
showed that these unknown factors induce cytoskeletal
and membrane rearrangements similar to those mediated
by either Zipper or Trigger mechanisms (Rosselin et al.,
2011). The signaling cascade induced by these unknown
factors seems specific because Aiastui et al. (2010) have
shown that Salmonella entry into immortalized human
foreskin fibroblasts did not require any of Rho GTPases
(Rac1, Cdc42, RhoA, or RhoG) involved in Rck and
T3SS-1 invasion pathways. Besides, for S. Typhimurium,
a novel ruffling-independent invasion pathway that could
operate independently of Arp2/3 complex has recently
been described. This entry mechanism relies on formation
of myosin II-rich stress fiber-like structures at invasion
sites through the activation of RhoA/Rho kinase signaling
pathway (Steffen et al., 2004; Hanisch et al., 2012).
Overall, these observations open up new perspectives for
identification of new invasion factors. Until now, it is not
clear why Salmonella serotypes use multiple strategies to
invade host cells. One possible explanation is that the
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
4 Z. Boumart et al.
development of alternate entry mechanisms mediated by
different invasins has broadened the spectrum of host cells,
which can be invaded by this intracellular pathogen.
II- Salmonella intracellular lifestyle:
Cytosolic vs. Vacuolar replication
In general, after eukaryotic cell invasion, bacteria are
internalized within a membrane-bound compartment.
Many bacterial pathogens have evolved different strategies
to counteract the host cell immune response, either by
preventing vacuole–lysosome fusion, modifying their vac-
uole or by escaping into cytosol. For example, Escherichia
coli modulates the maturation process of its vacuole to
avoid fusion with lysosomes (Kim et al., 2003), while Lis-
teria monocytogenes, Shigella flexneri, Francisella tularensis,
and some Mycobacterium species have the ability to
escape early from the nascent vacuole to enter the cyto-
plasm and exploit it as habitat for growth and replication
(Ray et al., 2009). Historically, Salmonella has been classi-
fied as a vacuolar pathogen. Upon invasion into host
cells, this bacterium resides and multiplies within the
SCV, a specialized vacuole that has been considered, for a
long time, as the only intracellular niche of Salmonella
(Bakowski et al., 2008). Nevertheless, recent evidence has
shown that a proportion of intracellular Salmonella can
escape from the SCV and replicates efficiently in the cyto-
sol of epithelial cells, indicating that this bacterium has
an intracellular bimodal lifestyle (Malik-Kale et al., 2012).
This phenomenon is, however, not observed in some cell
types such as macrophages, where cytosolic bacteria are
unable to survive or to grow because of the lethal envi-
ronment of the cytosol (Beuzon et al., 2002; Knodler
et al., 2010).
Following internalization in epithelial cells, the SCV
undergoes different stages of maturation similar to that of
macrophages. The newly formed SCV is enriched in
markers of early endosome such as Rab5, EEA1 (early
endosome antigen 1), and TfR (transferrin receptor),
which are subsequently replaced by late endosome and
lysosome markers, such as Rab7, LAMP-1 (lysosomal-
associated membrane protein 1), and V-ATPase that
allows the SCV acidification (pH 4–5). Some markers
such as Mannose-6-Phosphate Receptor (M6PR) involved
in the delivery of lysosomal hydrolases to the endosomal
system are not presented by the SCV to avoid fusion with
lysosomes (Steele-Mortimer et al., 1999; Bakowski et al.,
2008). As the SCV matures and is surrounded by actin, it
migrates toward a perinuclear position and initiates for-
mation of Salmonella-induced filaments (SIF), which
ensure delivery of nutrients to the SCV, thereby facilitat-
ing bacterial replication (Knodler & Steele-Mortimer,
2003; Salcedo & Holden, 2003).
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
In epithelial cells, a defect in vacuole maturation leads
sometimes to the release of Salmonella in the cell nutri-
ent-rich cytosol that can support a high bacterial replica-
tion rate (Brumell et al., 2002; Knodler et al., 2014).
It is now well documented that, the net intracellular
growth of Salmonella is the product of both vacuolar and
cytosolic replication and of intracellular bacteria destruc-
tion. The quantification of cytosolic Salmonella contribu-
tion to the total population in different epithelial cell
lines and under conditions of altered vacuolar escape has
shown that cytosolic Salmonella replicate at higher rate
than vacuolar bacteria, which has been termed hyper-
replication (estimated to ≥ 100 bacteria per cell) (Knodler
et al., 2014). Furthermore, it has been reported that cyto-
solic hyper-replication occurs in a minority (< 20%) of
infected epithelial cells (Malik-Kale et al., 2012). How-
ever, within fibroblast cells, very limited proliferation of
Salmonella has been described. It has been demonstrated
that Salmonella itself contributes to attenuating the intra-
cellular growth rate as bacterial virulence regulators, such
as PhoP/PhoQ system, are involved in preventing bacte-
rial overgrowth in the intracellular environment of pri-
mary fibroblasts (Cano et al., 2001). The escape of
Salmonella from the SCV and its hyper-replication in the
cytosol of epithelial cells is considered as a transition step
that precedes the bacteria exit into the extracellular
environment. Cytosolic Salmonella is sensed by the host
epithelial cells, leading to an inflammatory response char-
acterized by the activation of caspase 1 and caspases 3/7,
and the apical release of IL-18, an important cytokine
regulator of intestinal inflammation (Monack et al.,
2001). Dead cells are, thus, extruded out of the mono-
layer, releasing SPI-1-induced and flagellated Salmonella
(invasion-primed Salmonella) into the lumen of intestinal
tract, which facilitates infection of Salmonella to neigh-
boring cells and its dissemination in the host body or to
a new host (Cliffe et al., 2005; Knodler et al., 2010).
Studies assessing the role of Salmonella virulence
factors in the establishment of vacuolar and cytosolic
intracellular populations have shown that T3SS-1 effec-
tors, besides their role in invasion, are involved in early
stages of the SCV biogenesis (Steele-Mortimer et al.,
2002). In contrast, effectors injected by another T3SS,
encoded by the SPI-2 and called T3SS-2, contribute to
later SCV biogenesis including vacuolar maturation, bac-
terial replication, and SIF formation (Kuhle & Hensel,
2004; Figueira & Holden, 2012). In the SCV, when Sal-
monella adapts to the intravacuolar environment, the two
component regulatory system PhoP/PhoQ (1) represses
the expression of SPI-1 genes, that are not longer needed,
through reduction in hilA transcription; (2) increases
SPI-2 T3SS expression through binding to the ssrB pro-
moter and posttranscriptional regulation of SsrA (Bijlsma
FEMS Microbiol Lett 361 (2014) 1–7
Salmonella-host cell interaction
& Groisman, 2005; Golubeva et al., 2012). Furthermore,
it has been shown that PagN, activated by PhoP, is maxi-
mally expressed in the intravacuolar compartment having
an acidic pH and low Mg2+ concentration. Thus, it has
been postulated that PagN might be expressed at optimal
level when exiting from the SCV, macrophages, or epithe-
lial cells, allowing a subsequent interaction with neighbor-
ing cells until T3SS-1 is maximally expressed (Lambert &
Smith, 2008).
For Salmonella vacuolar escape, it has been shown that
only the T3SS-1, but neither the T3SS-2, nor flagella are
required. However, the cytosolic bacteria are flagellated
and express T3SS-1, but seem to not express the T3SS-2,
which could explain the delayed replication defect of
inactive T3SS-2 bacteria in epithelial cells (Malik-Kale
et al., 2012; Knodler et al., 2014).
Death of intracellular Salmonella could be related either
to the complete SCV–lysosome fusion (Viboud & Bliska,
2001), or to autophagy, a mechanism of capture of either
cytosol-adapted or vacuolar bacteria that redirect them to
the lysosomal compartment for killing. In fact, in some
cases, intravacuolar Salmonella can damage the SCV
membrane through T3SS-1 components (Roy et al.,
2004). The damaged SCV is targeted, as well as cytosolic
Salmonella, by the autophagy system, a mechanism
involved in the immune response against intracellular
pathogens. Salmonella is thus released in an autosomal
compartment that can affect its replication or induce its
destruction (Birmingham et al., 2006). However, many
studies have shown that, in some cases, Salmonella using
different strategies at different times could avoid host cell
response (lysosomal degradation and autophagy) and
ensure its infectious cycle. In epithelial cells, this autopha-
gic system showed to be not efficient against Salmonella
after escape from the SCV. This subpopulation of Salmo-
nella has, indeed, the ability to evade autophagic recogni-
tion in order to hyper-replicate in the host cell cytosol
(Knodler et al., 2014). Furthermore, a recent report has
shown that Salmonella benefits from autophagy for its
replication in the host cell cytosol. In HeLa cells, it has
been demonstrated that the lack of Rab1, a Rho GTPase
required for autophagy, significantly decreases Salmonella
replication and, contrary to intravacuolar bacteria, a pro-
portion of cytosolic Salmonella associated with autophagy
components p62 and/or LC3 can quickly replicate in the
host cell (Yu et al., 2014).
In addition, Salmonella, at later time points, may also
benefits from the SCV–lysosome fusion to proliferate in
the host cell without being destroyed. Indeed, Eswarappa
et al. have shown that in some cases, many vacuoles with
a single bacterium could be found inside one cell as a
result of the SCV division. The increase of the SCV num-
ber creates an imbalance in the ratio of the number of
FEMS Microbiol Lett 361 (2014) 1–7
5
vacuoles to the number of acidic lysosomes. Therefore,
having a cell with insufficient acidic lysosomes to fuse
with all the SCVs is advantageous for Salmonella by
favoring its survival and proliferation in the host cell
(Eswarappa et al., 2010).
Conclusion
Salmonella is a fascinating bacterium, which has evolved a
variety of strategies to invade and survive within the host
cells and thus become a successful pathogen. These recent
years, many molecular interactions between Salmonella and
the host cells have been elucidated, but many questions still
unanswered. Why Salmonella has developed alternate
mechanisms for cellular invasion? How this bacterium
could modify the SCV niche and adapt to the cytosolic life
style? What is the role of this new phenotype in Salmonella
pathogenicity? Do the entry mechanisms affect the intracel-
lular lifestyle of Salmonella and the host response induced?
It can be, indeed, hypothesized that the route of entry used
by Salmonella strains are different according to the serovar
and the host cell, which could modify the host range and/
or the pathogenicity of this bacterium. In addition, the
existence of synergy between the different invasion path-
ways used by Salmonella should not be excluded. There-
fore, further studies should be conducted on T3SS-1
independent mechanisms to identify the host receptors
involved and the signaling pathways induced. Further
investigation are also required to identify the bacterial fac-
tors involved in determining Salmonella intracellular locali-
zation and the role played by either cytosolic or vacuolar
proliferation in Salmonella pathogenesis.
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