Virulence Factors in Salmonella Typhimurium: The Sagacity of a Bacterium

Abstract

Currently, Salmonella enterica Typhimurium (ST) is responsible for most cases of food poisoning in several countries. It is characterized as a non-specific zoonotic bacterium that can infect both humans and animals and although most of the infections caused by this microorganism cause only a self-limiting gastroenteritis, some ST strains have been shown to be invasive, crossing the intestinal wall and reaching the systemic circulation. This unusual pathogenicity ability is closely related to ST virulence factors. This review aims to portray the main virulence factors in Salmonella Typhimurium, in order to better understand the strategies that this pathogen uses to reach the systemic circulation and increase its infectivity in humans and animals. Thus, the most studied Salmonella pathogenicity islands in Salmonella Typhimurium were detailed as to the functions of their encoded virulence factors. In addition, available knowledge on virulence plasmid was also compiled, as well as the chromosome regions involved in the virulence of this bacterium.

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Current Microbiology
https://doi.org/10.1007/s00284-018-1510-4
REVIEW ARTICLE
Virulence Factors inSalmonella Typhimurium: The Sagacity
ofaBacterium
AnamariaM.P.dosSantos1· RafaelaG.Ferrari1,2· CarlosA.Conte‑Junior1,2,3
Received: 4 October 2017 / Accepted: 16 May 2018
© Springer Science+Business Media, LLC, part of Springer Nature 2018
Abstract
Currently, Salmonella enterica Typhimurium (ST) is responsible for most cases of food poisoning in several countries. It is
characterized as a non-specific zoonotic bacterium that can infect both humans and animals and although most of the infec-
tions caused by this microorganism cause only a self-limiting gastroenteritis, some ST strains have been shown to be invasive,
crossing the intestinal wall and reaching the systemic circulation. This unusual pathogenicity ability is closely related to
ST virulence factors. This review aims to portray the main virulence factors in Salmonella Typhimurium, in order to better
understand the strategies that this pathogen uses to reach the systemic circulation and increase its infectivity in humans and
animals. Thus, the most studied Salmonella pathogenicity islands in Salmonella Typhimurium were detailed as to the func-
tions of their encoded virulence factors. In addition, available knowledge on virulence plasmid was also compiled, as well
as the chromosome regions involved in the virulence of this bacterium.
Introduction
Salmonella spp. represents one of the principal causes of
food poisoning in several countries in the last 100years [72].
Approximately 16million cases of typhoid fever, 1.3bil-
lion cases of gastroenteritis, and 3million deaths involving
this bacterium are reported annually worldwide [83]. Due
to its endemicity, high morbidity and, especially, difficulty
in applying control and prevention measures, salmonellosis
is characterized as an important zoonosis in a public health
context [90].
The genus Salmonella, belonging to the Enterobac-
teriaceae family, includes two species, S. enterica and S.
bongori [90]. The first comprises six subspecies designated
by Roman numerals [25], and includes more than 2600
serotypes that differ from each other by their flagellar (H)
and somatic (O) structures [17]. S. enterica subspecies I
(enterica) is the most isolated subspecies in animals, and
is found in 99% of human isolates. S. bongori, on the other
hand, is often found in “cold-blooded” animals, such as rep-
tiles, amphibians, and fish, and accounts for less than 1% of
human isolates [83].
Diseases related to Salmonella enterica can be divided
into three groups: Typhoid fever, caused by S. Typhi; enteric
fever, caused by S. Paratyphi A, B, and C; and enterocolitis
or salmonellosis, caused by the other serotypes [90]. Accord-
ing to Connor and Schwartz [19], because their symptoms
are indistinguishable in a clinical analysis, both S. Typhi and
S. Paratyphi are classified as causative agents for typhoid
fever. These bacteria have human and some larger primates
as their only reservoirs [33]. Symptoms are characterized
by abdominal pain, headaches, fever, and diarrhea that may
appear within a week of incubation [25]. In contrast, sal-
monellosis affects both human and animals and is the main
cause of gastroenteritis in humans [1], resulting in 93.8mil-
lion cases, with 155,000 deaths each year [25].
Among these bacteria, the most frequently isolated
serotypes are S. enterica Typhimurium (ST) and S.
enterica Enteritidis (SE) [35]. In Brazil, ST was the most
isolated serotype up to the mid-1990s, being surpassed
after this by SE [1]. ST is a general pathogen, and can
be isolated from different animals, as well as different
foodstuffs. Thus, this serotype can be found in a variety
of foods, and is mainly present in poultry [70, 103], swine
* Carlos A. Conte-Junior
carlosconte@id.uff.br
1 Department ofFood Technology, Faculty ofVeterinary,
Molecular & Analytical Laboratory Center, Universidade
Federal Fluminense, Niterói, Brazil
2 Chemistry Institute, Food Science Program, Universidade
Federal doRio de Janeiro, RiodeJaneiro, Brazil
3 National Institute ofHealth Quality Control, Fundação
Oswaldo Cruz, RiodeJaneiro, Brazil

A.M.P.dos Santos et al.
1 3
[75, 95], and bovine meat [84]. However, some ST strains,
in addition to causing localized gastroenteritis, are capa-
ble of causing systemic infections, such as the case of the
multi-locus sequence type 313 (ST313) isolated in sub-
Saharan Africa [61], and the most commonly found ST19
[12]. These bacteria multiply in the small intestine and
extend beyond the intestinal wall, reaching the mesenteric
lymph nodes and from there, the liver and spleen, where
they settle and multiply [44].
It is believed that such an ability was acquired from
horizontal gene transfer (transduction, conjugation, or
transformation) over millions of years [33]. An example
for this reasoning is the fact that ST and S. Typhi share
about 90% of the genes present in their DNA [57]. This
similarity allows ST to be used as a model for the study
of typhoid fever in animals, since S. Typhi is human-
specific [88]. The remaining 10% include virulence fac-
tors, defined as structures, products, and strategies that
contribute to infection and that determine the degree of
pathogenicity of this serotype [57]. In ST, most of the
genes that encode such factors are located in the so-called
Salmonella Pathogenicity Islands (SPI) [76]. Although
less relevant, they may also be present in other parts of
the chromosome, such as the fimbriae and flagella. The
genes encoding these characteristics can be found in viru-
lence plasmids (pSLT), more precisely in the spv operon
[26].
In this context, this review aims to portray the main
virulence factors in Salmonella Typhimurium in order to
better understand what strategies this pathogen uses to
reach the systemic circulation and increase its infectivity
in humans and animals (Table1).
Overview oftheInfection Process
Infection caused by ST occurs mainly through the inges-
tion of contaminated animal or water food [11, 76]. Upon
reaching the stomach, this microorganism must cope with
the acidity of the medium, so its acid tolerance response
(ATR) is activated, which maintains the intracellular pH
higher than the extracellular pH, allowing the bacterium to
survive while in this environment [26]. Subsequently, ST
crosses the mucus layer present in the intestinal wall and
adheres to the epithelium [7, 48], where the infection will
occur. The interaction of ST with the epithelium results in
the appearance of the clinical diagnosis, characterized by
diarrhea, culminating in the loss of electrolytes and inducing
local inflammation of the intestine [46].
ST adhesion to the epithelium is achieved by host–recep-
tor interactions with many of the adhesion factors present
on the cell surface of this microorganism [76]. After this
stage, effector proteins are released into the enterocyte cyto-
plasm, causing changes in the cytoskeleton of the intestinal
epithelium [76]. These modifications lead to the formation
of membrane extensions, known as ruffles, similar to those
formed by phagocytic cells [25]. The ruffles encompass ST
and launch it into the cell [11]. With the engulfment of the
bacterium by enterocytes, intracellular phagosomal compart-
ments, termed Salmonella-Containing Vacuoles (SCV), are
formed [24, 31, 44, 46, 50, 67] (Fig.1). This compartment
is the only place in these host cells where ST the can survive
and multiply [48]. As a consequence of this invasion, tran-
scription activators are generated, leading to the production
of proinflammatory cytokines, such as interleukin (IL)-8,
which ultimately induce the inflammatory response [46].
With this induction, polymorphonuclear leucocytes (PMN)
Table 1 Components of T3SS-1
and T3SS-2, substructures of
its components (if any), and
structural proteins that comprise
those components
Components Substructures Protein compo-
nents T3SS-1
Protein compo-
nents T3SS-2
References
Needle complex Needle structure PrgI SsaG [23, 60]
PrgJ SsaI
Needle complex Inner membrane rings PrgK SsaJ [23, 60]
PrgH SsaD
Outer membrane rings InvG SsaC
Export apparatus SpaO SsaQ [23, 60]
SpaP SsaR
SpaQ SsaS
SpaR SsaT
SpaS SsaU
InvA SsaV
InvC SsaN
OrgA –
Translocon SipBCD SseCDB [23, 60]

Virulence Factors inSalmonella Typhimurium: The Sagacity ofaBacterium
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migrate into the intestine and induce the production of anti-
microbial substances, where apparently ST is less affected
[52]. This generates a microenvironment in which ST has a
growth advantage over the resident microbiota, which is also
affected by the substances released by PMNs [7].
Jones etal. [56] noted that salmonellae adhere to and
preferentially invade M cells, which are modified cells of
the intestinal wall of the ileum present in Peyer’s plaques.
M cells are intimately associated with macrophages, which
reside in this tissue [53]. When the epithelial barrier is rup-
tured, ST can invade macrophages and dendritic cells and,
from these infected cells, gain systemic circulation [52].
During all stages of this infection, ST uses specific virulence
factors to conduct a more efficient invasion (Table2). In this
context, we will now report these factors and their roles in
infection caused by Salmonella Typhimurium.
Strategies andVirulence Factors
Type III Secretion System (T3SS)
Salmonella spp. possesses a molecular apparatus called the
Type III Secretion System (T3SS) [17, 44], which is respon-
sible for the ability of these microorganisms to inject effector
proteins into the cytosol of host cells and modulate host cells
signaling cascades for the benefit of bacteria [17, 57]. These
effectors, once within the cell, can alter cellular functions
such as cytoskeleton structure, membrane transport, signal
transduction, and cytokine expression [48]. These changes
allow for the invasion and permanence of the bacterium in
the infected cell [17]. T3SS comprises several components
(Table1), including more than twenty proteins [48], some of
them are homologous to those involved in flagellar assembly
[77], suggesting a evolutionary relation [66].
Salmonella enterica abstract two distinct T3SSs (T3SS-1
and T3SS-2), which are encoded by SPI-1 and SPI-2, respec-
tively, located in different Salmonella DNA clusters [46].
They have shown to function at different times during the
infection [46]. Thereby, T3SS-1 is active with the contact
of the bacterium with the host cell membrane and translo-
cates effector proteins into its cytoplasm, while T3SS-2 is
active within the phagosome and translocates effectors into
the vacuolar space [48]. This ability could be very important
for systemic increase of ST in its host, once T3SS-2 secreted
genes are required for bacterial growth in macrophages [16,
51, 82]. Most of the effectors translocated by T3SS-1 are
encoded on SPI-1, but, as we will show in this article, some
of them can be encoded on SPI- 5 [46], as well as some also
can be encoded in other parts of ST DNA [77]. Just like
T3SS-1, T3SS-2 translocates effectors which are encoded in
other parts of ST DNA [99], despites most of the effectors
Fig. 1 Pathogenesis of Salmonella Typhimurium. a Salmonella
adheres to the intestinal epithelial and M cells using many of adhe-
sions factors present on its cell surface. b, c Effector proteins are
released into enterocyte causing changes on its cytoskeleton and
forming structures in its surface known as ruffles. d Alternatively, the
bacterial cells can be directly taken by dendritic cell from the sub-
mucosa. e Once inside cytoplasm, Salmonella cells are located into
SCV (Salmonella Containing Vacuoles), where it multiplies. f The
SCV transcytose to the basolateral membrane and release to the sub-
mucosa. g Bacteria is internalized within phagocytes and then located
again into SCV; this figure was based on the one illustrated in the
article [89]

A.M.P.dos Santos et al.
1 3
translocated for this system is encoded on SPI-2 [51].
Although T3SS-1 and T3SS-2 have similar structures and
perform the same function (translocate effectors proteins),
these systems are not identical themselves, since both have
different structural proteins and, as said, secrete different
effector proteins [99]. Their structural proteins, however,
share some homology (Table1), which makes their mecha-
nisms not seem significantly different [71]. It is believed,
though, that they are not independent one of another, once
mutations in T3SS-2 causes a significant reduction in the
expression of some genes of T3SS-1, which harms the
ability of the bacterium to invade epithelial cells [22]. In
view of its homology, we prefer to use T3SS-1 of ST as
an archetype to prevent confusion and since T3SS-1 of ST
is, till now, the most well-characterized system [20, 59, 60,
66, 67]. The corresponding components of T3SS-2 can be
found in Table1.
Kubori etal. [61], after observing the structure of T3SS-1
apparatus under optical microscopy, has found a supramo-
lecular structure of ST T3SS-1 apparatus called the Nee-
dle Complex (NC) [48]. The NC is, by definition, a hollow
structure composed by two pairs of rings that are anchored
Table 2 Main virulence factors, DNA location, and main functions
a SPI Salmonella pathogenicity island
b SIF Salmonella-induced filaments
c SCV Salmonella-containing vacuoles
Virulence factor Location Function Reference
SipA SPI-1aCytoskeleton rearrangement [104]
Chemotaxis [76]
SipB SPI-1 Translocation of effector proteins [30]
Macrophage apoptosis impairment [18]
SipC SPI-1 Chemotaxis [76]
Cytoskeleton rearrangement
SptP SPI-1 Suppression of innate immunity [14]
trr genes SPI-2 Production of tetrathionate reductase [26]
SpiC SPI-2 Translocation of effector proteins [28]
Survival within SCVc[98]
SseB SPI-2 Formation of macromolecular structures which serves as a translocon [70]
SseC SPI-2 Formation of macromolecular structures which serves as a translocon [70]
SseD SPI-2 Formation of macromolecular structures which serves as a translocon [70]
SseF SPI-2 SCV perinuclear migration [68, 69]
Microtubule aggregation
SIF formationb
SseG SPI-2 SCV perinuclear migration [68, 69]
Microtubule aggregation
SIF formation
MisL SPI-3 Long-term persistence [24]
MgtCB SPI-3 Survival within macrophages [5]
MarT SPI-3 Activation of MisL expression [85]
SiiE SPI-4 Adhesion to the epithelium [34]
SopB SPI-5 Prevents apoptosis of epithelial cells [64]
SigE SPI-5 Chaperone [21]
SpvR pSLT Regulation of spv genes [38]
SpvB pSLT Prevents actin polymerization [38]
SpvC pSLT Inhibits MAP kinase and immune signaling [91]
Type I Fimbrae Chromosome Adhesion to the epithelium [3]
SifA Chromosome SIF formation [4]
SCV maintenance
SseJ Chromosome SIF formation [69]
SopE Chromosome Induce membrane ruffling in cell cultures [47]
SopE2 Chromosome Induce membrane ruffling in cell cultures [47]

Virulence Factors inSalmonella Typhimurium: The Sagacity ofaBacterium
1 3
to the inner and the outer membranes of the bacterium, and
a needle-like structure that extends beyond the surface of
the bacterium [59] (Fig.2). A NC is composed of at least
five proteins components: PrgI, J, K, H, and InvG [60], each
one of them is required for the type III secretion and the
invasion of epithelial cells [59]. PrgI was demonstrated to
be component of the needle portion of the NC, since ST prgI
mutant strain did not present needle substructures, although
it did exhibit apparently normal bases [67]. Beyond that, the
same study has showed that PrgI is essential for ST entry
into host cells, once prgI mutant was completely defective
in its ability to invade cultured intestinal cells [67]. PrgJ is
required for PrgI secretion as well as the formation of the
needle structure, and it is also the minor component of the
needle itself [60]. PrgH and PrgK, on the other hand, interact
with each other to form the inner membrane rings of the NC
[59]. Lastly, InvG, which is a member of the secretin fam-
ily of protein [67], forms the outer membrane rings of the
NC, which are believed to permit the passage of exported
substrates across the outer membrane [60]. Housed within
the inner membrane rings of the NC base, there is a Type III
Export Apparatus which is required to assembly of the NC
and the secretion of effector proteins [94]. This apparatus is
believed to be composed by eight proteins (SpaO, P, Q, R, S,
InvA, C, and OrgA), which are conserved among all T3SS
[55]. A few sets of other proteins (as SipBCD, explained
in the following section) comprise a Translocon, which is
believed to be involved in the translocation of effector pro-
teins into the host cytoplasm by forming a translocation pore
in the host cellular membrane [48, 60].
Salmonella Pathogenicity Islands
Salmonella pathogenicity islands (SPIs) are chromosomal
regions that carry virulence genes that act as a compact and
distinct genetic unit known as an operon [77]. These regions
display a different composition from the rest of the chromo-
some, with the characteristic presence of a greater amount
of G+C (guanine and cytosine) when compared with the
other parts of the DNA [79]. To date, five SPIs have been
described in ST, with SPI-1 and SPI-2 being the most rec-
ognized and studied [2, 17, 25, 44, 77]. Although the other
three SPIs have not been studied as closely as the first two
[26], studies have reported the involvement of these islands
in Salmonella invasion of and survival within host cells [11].
SPI-1 is the most well-characterized island among the
five ST SPIs [26, 33, 77]. Genes encoded by this region are
said to be essential in the invasion stage through the intesti-
nal epithelium [77], since salmonellae SPI-1 mutants have
shown an attenuation for systemic invasion when inoculated
orally in rats. However, this does not occur when inoculation
is performed by the parenteral route [31]. This suggests that
these genes are closely related to the host’s internalization
of the bacterium and crucial for ST to penetrate the intes-
tinal wall and consolidate the invasion [31]. SipABCD and
SptP are the major effector proteins encoded by these genes,
released into the host cell through T3SS-1.
SipA is said to be one of the first proteins responsible
for the induction of cytoskeletal rearrangement of epithelial
cells, which facilitates bacteria entry into the host cell [104].
This has been confirmed by invitro studies with ST sipA
mutants, in which it was demonstrated that such a microor-
ganism was less effective in inducing actin-cytoskeletal reor-
ganization in HeLa cells, in the same way that they were pre-
vented from being internalized by these cells [104]. As with
SipA, SipC is also responsible for inducing actin-cytoskel-
etal rearrangements [76] and, alongside SipB, is involved
in T3SS-1 translocation of effector molecules (e.g., SptP)
into the host cell [18]. Studies conducted with sipB, sipC
sipD mutants have shown the inability of these strains to
stimulate any response in the T3SS-1-dependent cell [18]. In
addition, SipA and SipC also activate a signal transduction
Fig. 2 T3SS-1 structure and its components. A Translocon, which
forms a translocation pore in the host cell membrane and translo-
cates effector proteins into the host cytoplasm, formed by SipB, C,
and D proteins; a needle structure, which extends beyond the surface
of the bacterium, formed by PrgI and PrgJ proteins; an outer mem-
brane rings, formed by InvG, located in outer membrane; and an
inner membrane rings, formed by PrgK and PrgH, located in the inner
membrane. In addition, an Export Apparatus, housed within the inner
membrane rings, formed by the proteins summarized in Table1; this
figure was based on the one illustrated in the article [31]

A.M.P.dos Santos et al.
1 3
cascade that leads to PMN migration [76], creating a micro-
environment favorable for ST growth [7]. The role of SipB
is still not fully elucidated [76]; however, Hersh etal. [53]
describe its ability to induce macrophage apoptosis from the
activation of caspase-1, and stated that Salmonella-induced
macrophage cytotoxicity is SipB-dependent.
Another protein encoded by genes located in SPI-1
is SptP. This effector is a phosphotyrosine phosphatase
which, when injected into epithelial cells, alters the actin
cytoskeleton [30]. Nevertheless, as demonstrated by Kaniga
etal. [58], SptP is not required for host cell invasion, since
sptP ST mutant strains enter cultured epithelial cells and
macrophages with the same efficiency as wild-type strains.
Likewise, sptP mutants did not display the same efficacy in
colonizing the liver of infected mice compared to wild-type
strains [58]. More recently, Choi etal. [14] reported the role
of SptP in suppressing the innate immunity of the host, dem-
onstrating that it is able to prevent mast cell degranulation,
which may be an important mechanism of ST virulence.
SPI-2 is a pathogenicity island that shows great impor-
tance in the maintenance of its permanence in the host cell.
This chromosome sequence contains about 40 genes [77],
grouped into four operons: ssa (type 3 secretion system), ssr
(secretion system regulation), ssc (chaperone secretion), and
sse (effector coding) [51].
SPI-2 is divided into two segments, one large and one
small [26]. The latter is characterized by the presence of trr
genes, which are involved in the production of tetrathion-
ate reductase, responsible for Salmonella-like tetrathionate
respiration [50] and seven open reading frames (ORFs) [26],
which, to date, have not demonstrated a significant role in
ST virulence [26]. On the other hand, the major follow-up
displays some importance in the ability of ST to survive
and multiply within SCV in host cells, either epithelial or
macrophages [82]. Fields etal. [27] reported that salmonel-
lae deficient in macrophage replication display attenuated
virulence, corroborating the idea that this capacity is essen-
tial for Salmonella pathogenesis. Similarly, Cirillo etal. [16]
have shown that SPI-2 mutants can colonize Peyer’s plaques,
but are unable to colonize the liver, spleen, or mesenteric
lymph nodes, which require invasion into macrophages. This
ability is given, mainly, by effector proteins SpiC, SseF, and
SseG that are translocated by T3SS-2.
Uchiya etal. [98] reported the first protein encoded
by SPI-2 and translocated via T3SS-2 to the macrophage
cytoplasm, SpiC. In their study, SpiC was classified as the
protein responsible for the inhibition of phagolysosome for-
mation (junction of lysosome-containing vacuoles with the
SCV). This mechanism enabled ST to be able to survive for
more than 24h within macrophages and dendritic cells [45].
Uchiya etal. [98] also demonstrated that spiC is of great
importance for ST virulence, since its deletion resulted in a
considerable attenuation of this characteristic. According to
Freeman etal. [28], SpiC is still required for the transloca-
tion of effector proteins inside host cells, such as SseB and
SseC.
Effector proteins SseB, SseC, and SseD have been shown
to be secreted by T3SS-2 when ST is cultured invitro under
SPI-2-inducing conditions [63]. These same proteins are
found on the cell surface of the bacterium and are necessary
for translocation of other effector proteins to the infected cell
[80]. They are believed to form a macromolecular structure
in the membrane that serves as a translocon [80]. In addi-
tion, SseC and SseD have also been shown to be essential for
virulence, since sseC and sseD mutants display a decrease
in this characteristic [63].
Hensel etal. [51], when analyzing sseF and sseG mutants,
noted that these genes play a role in systemic pathogenesis
and intracellular proliferation. In addition, Kuhle and Hensel
[68] demonstrated that sseF and sseG may be related to the
induction of Salmonella-induced filaments (SIF), since their
exclusion leads to a considerable decrease in the formation
of these structures when compared to wild-type strains. SIF
are structures that arise when, a few hours after internaliza-
tion, SCV begin to elongate into tubular filaments [8]. Their
formation involves the fusion of late endocytic compart-
ments [10] and, although this is clear, the mechanisms that
mediate this process are still unknown [8]. Studies, how-
ever, suggest that microtubules serve as a support for such a
formation [69]. Finally, Kuhle etal. [69] demonstrated that
SseF and SseG are co-localized with microtubules, present
in endosomal compartments that aggregate to SIF along
these same structures, further corroborating the hypothesis
postulated by Kuhle and Hensel [68].
As mentioned previously, the other SPIs has not been
described in as much detail as the first two, although some
information is available. SPI-3, for example, contains ten
ORFs organized into six transcriptional units [77]. The
proteins encoded by these genes, however, play roles with
no obvious relation to each other [6]. mgtCB, for exam-
ple, encodes proteins that are required for survival within
macrophages [5], thus contributing to its virulence. On the
other hand, MisL, a protein encoded in SPI-3, contributes to
intestinal colonization and is required for long-term intesti-
nal persistence and involved in host–pathogen interactions
during infection [24]. In addition, MarT has demonstrated
regulatory functions [26], in addition to activating MisL
expression [97].
SPI-4 contains six ORFs arranged in a single operon,
called siiABCDEF [26]. Morgan etal. [78] demonstrated
that, although not necessary for systemic infection, SPI-4
is required for intestinal colonization. Taking this into
consideration, Gerlach etal. [34] analyzed the role of SPI-
4-encoded proteins in the interaction with epithelial cells
and demonstrated that this island plays an important role in
adhesion to the intestinal epithelium. By infecting epithelial

Virulence Factors inSalmonella Typhimurium: The Sagacity ofaBacterium
1 3
cell lines (MDCK) with wild-type Salmonella and SPI-4-de-
ficient strains, Gerlach etal. [34] observed that a considera-
ble percentage (4.24%) of the wild-type Salmonella adhered
to the epithelium, whereas only 0.28% of SPI-4-deficient
Salmonella displayed this characteristic. Regarding the
SiiE protein encoded by the sii operon, some non-fimbrial
adhesin properties are displayed, evidenced by decreases
in adhesion when the siiE gene is deleted [34]. In order to
discover the individual role of each gene encoded by the sii
operon, Kiss etal. [62] tested the virulence of mutants carry-
ing non-polar deletions in individual sii genes with wild-type
Salmonella strains. The results demonstrated that, even at
different levels, wild-type strains were more virulent than
the six sii mutants, suggesting that they all display some sig-
nificance in Salmonella virulence. However, further studies
should be conducted to find out the exact role of these genes
in ST pathogenicity.
SPI-5 is composed of five genes (pipD, sigD/sopB,
sigE/pipC, pipB, and pipA) [64], which may be related to
the role of SPI-5 in enteropathogens [77]. Among the five
coded proteins, SopB (SigD in ST) is the best described and
known to be translocated by T3SS-1 into the host cell cyto-
plasm [46]. Galyov etal. [32] demonstrated that, although
sopB deletion did not affect pathogen invasiveness, SopB
plays an important role in the induction of fluid secretion by
enterocytes, as well as in PMN induction in the intestine. In
addition, SopB also presents inositol phosphate phosphatase
activity, which is directly related to the induction of diarrhea
[81]. Knodler etal. [65], on the other hand, demonstrated
that SopB is able to prevent apoptosis of cultured epithe-
lial cells from the activation of Akt, an important apoptosis
regulator in these cells, in a mechanism used by the host to
protect itself from a pathogen. SigE was characterized by
Hong and Miller [54] as a possible chaperone that acts on
the stability of SopB secretion. This hypothesis was later
confirmed by Darwin etal. [21]. Finally, mutational studies
were performed by Wood etal. [101] in order to discover the
role of the pipA, pipB, and pipD genes. None of the mutants
in these genes had their growth rate or ability to invade HeLa
cells affected. However, both inflammatory responses and
the rate of fluid secretion were reduced in pip mutants com-
pared to wild-type strains. This suggests that these genes
are directly linked to the Salmonella enteropathogenicity,
although they do not have an effect on the development of
systemic invasion [101].
Virulence Plasmids
Several Salmonella enterica carry virulence plasmids,
including S. Enteritidis, S. Dublin, S. Choleraesuis, and S.
Typhimurium [39–41]. Such plasmids are differentiated
among serotypes according to their size [15]. In ST, the viru-
lence plasmid contains 95kb size [15] and is named pSLT
[92, 96]. It is believed that this structure was acquired hori-
zontally, since it is located in a region adjacent to the inser-
tion element and contains 46% less G+C than other parts
of the chromosome [77]. pSLT is equipped with a highly
conserved 8-kb sequence, with five ORFs, named the spv
operon (Salmonella plasmid of virulence) [40]. This small
part of the plasmid, however small, can restore virulence
in plasmid-cured salmonellae in a mouse model [41]. This
information led several research groups to further study the
role of spv genes in virulence, since strains lacking the spv
operon proved to be avirulent [39]. Gulig and Doyle [42],
for example, observed that spv genes were directly related to
the ability of ST to increase its multiplication rate within the
host during infection. Later, Guilloteau etal. [36] analyzed
the interactions of many wild-type and plasmid-cured Sal-
monella serotypes invitro murine and bovine macrophages
and invivo mice after infection. The presence of virulence
plasmids increased the lytic activity in macrophages infected
by ST, S. Dublin, and S. Choleraesuis, besides inducing
inflammatory responses in the peritoneum. Gulig etal. [43],
on the other hand, examined the relationship between spv
genes and the rate of ST growth in different cell populations,
including lymphocytes and neutrophils in addition to mac-
rophages. Genetic markers were used to measure the number
of cell divisions in cells infected with ST strains with or
without the virulence plasmid. It was noted that only the
macrophages showed relevance for ST multiplication [43],
since, with the quantitative decrease of these cells, the two
treatments (with and without the plasmid) showed the same
results for virulence [43]. Recently, Wu etal. [102], based
on analyses in zebrafish larvae, demonstrated the ability of
wild-type strains of ST to inhibit the autophagic activity of
macrophages and neutrophils, in addition to repressing the
function of macrophages and neutrophils when compared
to ST spv mutants. Thus, they demonstrated that the spv
genes are also involved in the suppression of innate immune
response of the host [102]. All these studies suggest the
important role that the spv operon plays in ST virulence,
although the mechanisms it applies are not yet fully under-
stood [102].
However, some mechanisms of effector proteins encoded
by spv operon have been elucidated. As mentioned previ-
ously, spv operon consists of five genes. The first one is spvR,
which is also the first gene to be transcribed [41]. The effec-
tor protein it encodes, SpvR, has as main function regulat-
ing the expression of the other four genes [40]. This protein
displays homology with MetR, a LysR member of positive
regulatory proteins [37]. SpvB is a cytotoxic protein [26],
which has the function of avoiding, above all, actin polym-
erization [38], being thus related to the intracellular phase
of infection. Additionally, Li etal. [74] demonstrated the
role of spvB in inhibiting autophagy in a zebrafish infection
model. This mechanism results in an increase of Salmonella

A.M.P.dos Santos et al.
1 3
virulence, since autophagy is a natural defense mechanism
of organisms infected by this pathogen [74]. SpvB was also
shown to be indispensable for virulence, since spvB mutants
are avirulent in mice [86]. SpvC is an anti-inflammatory
effector which plays an important role in the host’s proin-
flammatory response [26], since it inhibits MAP kinases
and, consequently, immune signaling [91]. Until recently,
SpvD still did not have its role defined; however, recently
Rolhion etal. [85] demonstrated the importance of this pro-
tein in the suppression of the immune response, interfering
with the transport of nuclear machinery mediated by NF-κβ.
Finally, SpvA still has no elucidated role in ST virulence,
requiring further studies [86].
Fimbriae andFlagella
Fimbriae are structures found on the cell surface of some
bacteria, which have been shown to play an important role in
the formation of biofilms, colonization, and the initial attack
of the bacterium on the host [57]. With the sequencing of the
ST genome, 13 operons were discovered [agf (csg), fim, lpf,
pef, bcf, stb, stc, std, stf, sth, sti, saf, and stj] with homology
to fimbrial biosynthesis genes [26, 76]. Since then, several
studies have been carried out to determine their action on the
virulence of this pathogen. Bäumler etal. [3] used a genetic
approach to investigate the role of three fimbrial operons
(fim, lpf, and pef) in ST adhesion to different epithelial cell
lines (HEp-2 and HeLa). These operons encode type I fim-
briae, long polar fimbriae, and plasmid-encoded fimbriae,
respectively [3]. The results showed a significant decrease in
adhesion to HEp-2 cells only for lpf ST mutants, whereas the
end deletion significantly decreased ST adhesion to HeLa.
These results suggest that the bacterial repertoire of fimbrial
adhesins determines which type of epithelial cell the bacte-
rium will adhere to during ST intestine infection [3]. Ween-
ing etal. [100] tested the role of colonization of ten fimbrial
operons (fim, pef, lbf, bcf, stb, stc, std, stf, sth, and agf) of
the bacteria in the intestine. Genetically resistant mice (e.g.,
CBA) were submitted to fimbrial operon mutation strains
and the results demonstrated that, from fimbrial operated
operons, only bcf, lpf, stb, stc, std, and sth contribute to the
ability of ST to be shed with the feces and to colonize the
cecum after 30 days of infection [100]. However, these same
data demonstrated that persistence in the intestine is com-
plex and involves the expression of more than 32 ST genes
[100]. Although we know the importance of these operons
in virulence, the exact mechanisms remain to be elucidated.
The flagella is a long helical filament coupled to rotat-
ing motors embedded within the outer membrane and cell
wall, which enables ST and the other bacteria that display
this feature to mobilize through the epithelial barrier after
ingestion [57]. It is characterized as a strong inflammation
inducer, mainly from the induction of Interleukin (IL)-8 and
activation of NF-κβ [17], a protein complex that plays the
role of a transcription factor and is involved in the immune
response to infection [73]. This induction is due to its chem-
otactic potential, which is also one of the main flagella char-
acteristics [13, 26].
Factors Present inOther Parts oftheChromosome
Some effector proteins are encoded outside the SPIs and
translocated to the host cell by T3SS. SopE (located in a
cryptic prophage) and SopE2, for example, although not
encoded in SPI-1, are translocated by T3SS-1 [77]. These,
together with the Sip proteins, play an important role in ST
invasion. Studies have demonstrated the ability of SopE
to induce membrane ruffling of cell cultures and that this
induction occurs from the GDP/GTP nucleotide exchange
in the Rho family, the G protein family responsible for the
intracellular dynamics of actin [49]. Because they are about
70% identical [47], SopE and SopE2 have similar roles in the
invasion [46]. However, Friebel etal. [29] demonstrated that
SopE and SopE2 activate different sets of RhoGTPases in
the host cell, suggesting that this small difference is respon-
sible for the existence of such similar proteins in the same
bacterium.
Other important effector proteins for ST virulence are
SifA and SseJ. These effectors are translocated into the host
cell cytoplasm by T3SS-2 [99]. They seem to have some
relation to each other [87] and their roles in ST virulence
are similar. Studies in sifA [4, 9] and sseJ [87] mutants dem-
onstrated an attenuation in their replication in macrophages
when compared with wild-type strains. In addition, a few
hours after internalization, sifA mutants appeared to dis-
play vacuolar membrane loss, with these organelles being
found loose in the host cell cytosol [4]. This demonstrates
the importance of this protein in both SCV maintenance and
intracellular replication. Another study reported that SifA is
also required for the formation of SIF [93] and, later, Kuhle
etal. [69] reached the same conclusion for SseJ, demonstrat-
ing that SseJ, like SseG and SseF, is located along with the
microtubules in host cells, which, as mentioned previously,
serve as support for the formation of SIF.
Conclusions
Salmonella Typhimurium is an intracellular pathogen, and
its ability to invade host cells and disseminate in the body is
closely related to its virulence genes. Further study of such
genes may allow for therapeutic measures against this micro-
organism to be developed, since the general knowledge of the
mechanisms used by ST in the invasion puts us one step ahead
in the fight against it. In light of what has been reported in this
review, it is notable that several advances have been achieved

Virulence Factors inSalmonella Typhimurium: The Sagacity ofaBacterium
1 3
in the research on the main factors of ST virulence. Among
these, it is perceived that SPIs play a crucial role in ST patho-
genicity, being, therefore, the most studied. The main studies
about the virulence of this pathogen are in the SPI-1 and SPI-
2. The discovery that both encode T3SSs was of great value,
since these apparatuses have the ability to inject not only effec-
tor proteins encoded in SPI-1 and 2, but also those encoded in
other parts of the chromosome into the cytoplasm of the host
cell. This fact further heightens the importance of these two
islands in maintaining virulence, since their absence eliminates
the ability of ST to inject T3SS-dependent effector proteins.
Despite this, much evidence also points out the role of the
other three SPIs, SPI-3, 4, and 5 on ST virulence. However,
much of the knowledge about these islands is still based on
empirical evidence, due to the absence of theoretical-scientific
basis, which can only be acquired with more research in the
area. An example of such structures are the SPI-4 sii genes and
Pip proteins in SPI-5, in which the exact mechanisms behind
the fact that their causes a certain attenuation in ST virulence
are not yet fully elucidated.
On the other hand, it has been shown that pSLT is
required for ST virulence. However, some mechanisms of
certain proteins produced in the spv operon, such as SpvA,
as well as their role in virulence, have not yet been fully elu-
cidated, requiring more comprehensive studies. Likewise, 13
operons encoding fimbriae are also involved in ST virulence,
contributing mainly to the adhesion of this bacterium to the
host cell. However, the exact mechanisms used by each gene
comprising this operon have not yet been discovered. Thus,
it can be concluded that, although many advances have been
made, there are still many challenges to be overcome regard-
ing ST virulence factors.
Acknowledgements We thank Virgínia P. Silveira for the design of
the figures.
Funding This study was funded by Fundação de Amparo à Pesquisa
do Estado do Rio de Janeiro (Process No. 232227, FAPERJ, Brazil).
Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (pro-
cess no. E-26/201.185/2014, FAPERJ, Brazil); Conselho Nacional de
Desenvolvimento Científico e Tecnológico (Process No. 311422/2016-
0, CNPq, Brazil), and Coordenação de Aperfeiçoamento de Pessoal
de Nível Superior (Process No. 125, CAPES/Embrapa 2014, CAPES,
Brazil).
Compliance with Ethical Standards
Conflict of interest The authors declare that the research was con-
ducted in the absence of any commercial or financial relationships that
could be construed as a potential conflict of interest.
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