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Host–Pathogen Interaction in Invasive Salmonellosis

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Host–Pathogen Interaction in Invasive Salmonellosis

Hanna K. de Jong1,2, Chris M. Parry3,4, Tom van der Poll1,2,5, W. Joost Wiersinga1,2,5*
1 Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center, Amsterdam, the Netherlands, 2 Center for Experimental and Molecular Medicine
(CEMM), Academic Medical Center, Amsterdam, the Netherlands, 3 Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University,
Bangkok, Thailand, 4 Centre for Tropical Medicine, Nuffield Department of Clinical Medicine, Churchill Hospital, University of Oxford, Oxford, United Kingdom,
5 Department of Medicine, Division of Infectious Diseases, Academic Medical Center, Amsterdam, the Netherlands

disease [2]. S. Typhi is an exclusively human pathogen causing a

Abstract: Salmonella enterica infections result in diverse bacteremic disease that, unlike many other Gram-negative
clinical manifestations. Typhoid fever, caused by S. bacteremias, does not typically manifest with neutrophilia or
enterica serovar Typhi (S. Typhi) and S. Paratyphi A, is a septic shock [3]. The widespread appearance of antimicrobial-
bacteremic illness but whose clinical features differ from resistant strains has limited treatment options [4,5]. Relapse and
other Gram-negative bacteremias. Non-typhoidal Salmo- chronic asymptomatic fecal carriage may complicate the illness
nella (NTS) serovars cause self-limiting diarrhea with
(Figure 1) [6,7]. Mortality usually results from intestinal perfora-
occasional secondary bacteremia. Primary NTS bacteremia
can occur in the immunocompromised host and infants in tion and peritonitis or from a severe toxic encephalopathy
sub-Saharan Africa. Recent studies on host–pathogen associated with myocarditis and hemodynamic shock [8].
interactions in Salmonellosis using genome sequencing, Infections with non-typhoidal Salmonella (NTS) serovars, such as
murine models, and patient studies have provided new S. enterica serovar Typhimurium and S. Enteriditis, also cause a
insights. The full genome sequences of numerous S. significant disease burden, with an estimated 93.8 million cases
enterica serovars have been determined. The S. Typhi worldwide and 155,000 deaths each year (see [9] for review) [10].
genome, compared to that of S. Typhimurium, harbors NTS serovars usually cause self-limiting diarrhea with secondary
many inactivated or disrupted genes. This can partly bacteremia occurring in less than 10% of patients. The host range
explain the different immune responses both serovars of non-typhoidal Salmonella serovars is broad, including poultry and
induce upon entering their host. Similar genome degra- cattle, and NTS infection is commonly due to food poisoning in
dation is also observed in the ST313 S. Typhimurium strain developed countries. NTS serovars cause high rates of bacteremia
implicated in invasive infection in sub-Saharan Africa. in the immunocompromised and, in sub-Saharan Africa, in
Virulence factors, most notably, type III secretion systems, children below 5 years old and those with HIV infection [9,11].
Vi antigen, lipopolysaccharide and other surface polysac-
Antimicrobial resistance is widespread [12].
charides, flagella, and various factors essential for the
intracellular life cycle of S. enterica have been character- The variations in the clinical features of infection with this
ized. Genes for these factors are commonly carried on intracellular pathogen relate to differences in the interaction
Salmonella Pathogenicity Islands (SPIs). Plasmids also carry between different Salmonella serovars and the host. This review
putative virulence-associated genes as well as those summarizes new and significant insights concerning the virulence
responsible for antimicrobial resistance. The interaction factors of both typhoid and non-typhoidal Salmonellae, their
of Salmonella pathogen-associated molecular patterns difference at the genome level, novel mechanisms employed by
(PAMPs) with Toll-like receptors (TLRs) and NOD-like these intruders to circumvent the host defense, and their
receptors (NLRs) leads to inflammasome formation, interactions with both host microbiome and the innate immune
activation, and recruitment of neutrophils and macro- system.
phages and the production of pro-inflammatory cyto-
kines, most notably interleukin (IL)-6, IL-1b, tumor necrosis
factor (TNF)-a, and interferon-gamma (IFN)-c. The gut
microbiome may be an important modulator of this
immune response. S. Typhimurium usually causes a local
intestinal immune response, whereas S. Typhi, by Citation: de Jong HK, Parry CM, van der Poll T, Wiersinga WJ (2012) Host–
preventing neutrophil attraction resulting from activation Pathogen Interaction in Invasive Salmonellosis. PLoS Pathog 8(10): e1002933.
of TLRs, evades the local response and causes systemic doi:10.1371/journal.ppat.1002933
infection. Potential new therapeutic strategies may lead Editor: Chetan E. Chitnis, International Centre for Genetic Engineering and
from an increased understanding of infection pathogen- Biotechnology, India
esis. Published October 4, 2012
Copyright: ß 2012 de Jong et al. This is an open-access article distributed
under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the
original author and source are credited.
Introduction
Funding: W.J. Wiersinga is supported by the Netherlands Organisation of
Typhoid fever is a global problem, with more than 27 million Scientific Research (VENI grant) and the Netherlands Organisation for Health
Research and Development (Clinical Fellowship grant). C.M. Parry is supported by
cases worldwide each year resulting in an estimated 217,000 the Wellcome Trust as part of the Wellcome Trust–Mahidol University–Oxford
deaths [1]. Salmonella enterica serovar Typhi (S. Typhi) and S. Tropical Medicine Research Programme. The funders had no role in study design,
Paratyphi A are the Gram-negative bacteria that cause this data collection and analysis, decision to publish, or preparation of the manuscript.
debilitating condition. It is most common among children, Competing Interests: The authors have declared that no competing interests
especially in areas of Asia and Africa that lack clean water and exist.
adequate sanitation, and is also an important travel-associated * E-mail: w.j.wiersinga@amc.uva.nl

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Figure 1. Dissemination of S. Typhi during systemic infection. Typhoid is usually contracted by ingestion of food or water contaminated by
fecal or urinary carriers excreting S. Typhi. The incubation period is usually 7 to 14 d. In the small intestine the bacteria adhere to the mucosa and then
invade the epithelial cells. The Peyer’s patches, which are aggregrated lymphoid nodules of the terminal ileum, play an important role in the transport
to the underlying lymphoid tissue. Specialized epithelial cells such as M cells overlying these Peyer’s patches are probably the site of internalization of
S. Typhi. Once the bacteria have penetrated the mucosal barrier, the invading organism translocates to the intestinal lymphoid follicles and the
draining mesenteric lymph nodes, and some pass on to the reticuloendothelial cells of the liver and spleen. During the bacteremic phase, the bacteria
are widely disseminated throughout the body. Secondary infection can occur with liver, spleen, bone-marrow, gallbladder, and Peyer’s patches as the
most preferred sites. The gallbladder is the main reservoir during a chronic infection with S. Typhi and invasion occurs either directly from the blood
or by retrograde spread from the bile. Of interest, the ability of Salmonella to form biofilms on gallstones is likely to be a critical factor in
establishment of chronic carriage and shedding of S. Typhi [88]. The bacteria that are excreted in the bile can then reinvade the intestinal wall by the
mechanism previously described or are excreted by feces. Typical clinical symptoms are fever, malaise, and abdominal discomfort. Clinical features
such as a tender abdomen, hepatomegaly, splenomegaly, and a relative bradycardia are common. Rose spots, the classical skin lesions associated
with typhoid fever, are relatively uncommon and occur in 5%–30% of cases. The most severe manifestations of typhoid leading to sepsis and death
are either necrosis of the Peyer’s patches resulting in gut perforation and peritonitis or a toxic encephalopathy associated with myocarditis and
haemodynamic shock [8,89].
doi:10.1371/journal.ppat.1002933.g001

The Bacteria ago [17]. The sequence-based technique of MLST (multilocus

sequence typing) provides a more accurate indication of the
Taxonomy and Genomics of Salmonella genomic relationship between different Salmonella isolates and may
The genus Salmonella is composed of two distinct species: supersede serotyping in the future [18]. Of the ,4,000 S. Typhi
Salmonella bongori and Salmonella enterica, the latter being divided into genes, more than 200 are functionally disrupted or inactive, while
six subspecies. These subspecies are classified into more than 50 most of these homologs are still fully functional in S. Typhimur-
serogroups based on the O (somatic) antigen, and divided into ium. This could in part explain the restricted host range of S.
.2,400 serovars based on the H (flagellar) antigen. Complete Typhi [16]. Although it has been suggested that the different
genome sequence from multiple Salmonella strains are available clinical outcomes of infection between typhoid and NTS serovars
[13]. For example, the S. Typhi type strain Ty2, the multidrug- may be explained by differences in genome expression leading to
resistant (MDR) isolate CT18, and the S. Typhimurium strain differences in host-pathogen recognition, one should also consider
LT2 are composed of 4.79 (Ty2), 4.86 (CT18), and 4.81 (LT2) the opposite possibility; that is, differences in host-pathogen
megabases, respectively [14–16]. The core genomes of Escherichia interaction may make certain genes dispensable, resulting in the
coli and S. enterica differ by only 10% in their DNA sequences and accumulation of pseudogenes [3,19,20].
suggest that the two species derived from a common ancestor Recent analysis of S. Typhimurium isolates from the unusually
about 100 million years ago. Comparison of different S. Typhi invasive infections seen in sub-Saharan Africa have shown
isolates show that they are highly related (clonal) and have arisen dominance of a particular MLST type, ST313, distinct from the
from a single point of origin approximately 30,000–50,000 years usual S. Typhimurium sequence type, ST19, associated in other

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parts of the world with diarrhea. In the D23580 invasive S. the spv genes. The spvB enzyme, which acts as an intracellular
Typhimurum isolate from Malawi, there is loss of gene function, ADP-ribosylating toxin causing host cytotoxicity, is necessary for
including genes previously implicated in the virulence of S. intra-macrophage survival but is absent in S. Typhi and S.
Typhimurium in the murine model of infection, such as sseI Paratyphi A [20,23]. Putative virulence-associated plasmid has
(encoding a type III-secreted effector protein) and ratB (encoding a only recently been identified in S. Typhi. The chimeric plasmid
secreted protein associated with intestinal persistence), and of the pR(ST98) carries genes that are involved in drug resistance and
44 novel pseudogenes or deletions in the strain relative to LT2, 26 apoptosis induction in macrophages [24,25]. Additionally, a linear
are also pseudogenes or deletions in S. Typhi or S. Paratyphi A plasmid in S. Typhi strains originating from Indonesia, called
[21]. These observations suggest that a similar process of adaption pBSSB1, carries the fljBz66 gene, which encodes a flagellin antigen
to the human host may be occurring in African S. Typhimurium as known as H:z66 [26]. Whether the presence of these particular
has been observed in S. Typhi. plasmids has an impact on the virulence of S. Typhi is not known.
The role of plasmids carrying antimicrobial resistance genes,
Virulence Factors such as cat, dhfr7, dhfr14, sul1, and blaTEM-1, in the transfer and
About 90% of the genes in S. Typhi and S. Typhimurium spread of antimicrobial resistance has been well described [20,27].
serovars are identical [16]. The 10% of genes that differ include S. Typhi is able to exchange multidrug resistance R-plasmids with
virulence factors, which determine their pathogenic potential E. coli and other enteric bacteria [28,29]. The self-transmissible
(Figure 2) [20]. The virulence factors of the Salmonella serovars are incompatibility group (Inc)HI1 plasmids almost exclusively confer
mostly encoded on the Salmonella pathogenicity islands (SPI). the phenotype of MDR S. Typhi. The presence of the MDR
Plasmids and prophages. Integrated bacteriophages, phage phenotype has been suggested to be associated with the
remnants, or plasmids are single- or double-stranded DNA development of severe or fatal disease [30,31]. The presence of
molecules that can be exchanged between bacteria by horizontal a composite genetic element encoding multiple antimicrobial
gene transfer. They give bacteria the opportunity to pass on or resistance genes on the virulence-associated plasmid in the ST313
receive selected genes that may enhance virulence or result in serovar Typhimurium isolates causing invasive disease in Africa
antimicrobial resistance [17,22]. Certain Salmonella spp. have a may provide an explanation for a linkage between drug resistance
self-transmissible virulence plasmid called pSLT, which harbors and an invasive phenotype [21]. Prophages and phage remnants

Figure 2. Virulence of S. Typhimurium and S. Typhi. S. Typhimurium and S. Typhi possess partly overlapping and a partly distinct repertoire of
virulence factors. Both serovars express the type III secretion system, lipopolysaccharide, and other surface polysaccharides, fimbrae, flagellin, and
bacterial DNA. The Vi antigen is exclusively expressed by S. Typhi and is able to circumvent the innate immune response by repressing flagellin and
LPS expression. SPI, Salmonella pathogenicity islands.
doi:10.1371/journal.ppat.1002933.g002

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can carry non-essential ‘‘cargo’’ genes involved in bacterial inflammatory responses [39]. However, S. Typhimurium mutants
virulence including several type III secretion system effectors, deficient in SPI-1 can disseminate and cause systemic infection
which play an important role in Salmonella virulence [20,32]. from the gastrointestinal tract by CD-18 expressing phagocytic
Type III secretion system and outer membrane cells such as dendritic cells (DCs) without disrupting the epithelial
vesicles. Salmonella enterica spp. contains two type III secretion barrier [40,41]. Therefore, this alternative pathway via CD-18
system (T3SS) gene clusters encoding a secretion apparatus that immune cells facilitates the development of a systemic infection for
functions like a molecular syringe. The T3SS secretes effector intracellular-adapted bacteria like Salmonella. T3SS2, encoded on
proteins into the target-cell cytosol, which manipulate host-cell SPI-2, plays a crucial role during the second phase of invasion,
signaling cascades. These effector proteins are suggested to have intracellular survival in macrophages [42]. Within the phagocyte,
multiple activities within host cells; for example, SopB is involved T3SS2 prevents trafficking from the phagocyte NADPH oxidase
in invasion and Akt activation, which causes fluid secretion and (nicotinamide adenine dinucleotide phosphate-oxidase) towards
Salmonella containing vacuole (SCV) formation (Figure 3a) [33–36]. the SCV, thereby preventing a phagocytic burst [43,44]. Although
The majority of genes encoding for these virulence-associated the importance of these systems for the virulence of S.
effector molecules are located on the SPIs. Other effector proteins Typhimurium is clear, limited data are available concerning the
such as SopE, SspH1, SseI, SodC-1, and SopE2 are encoded by role of T3SS in S. Typhi. Of note, the SPI-2 T3SS of S. Typhi is
phages or phage remnants [22,37,38]. not required for survival in human macrophages but may be used
Of the 21 SPIs known to date, SPI-1 and SPI-2 are the most during infection of other cell types, such as DCs or natural killer
studied. S. Typhimurium and S. Typhi genomes share 11 common cells, leading to the notion that the SPI-2 T3SS may be required to
SPIs; four are specific to S. Typhi (SPI-7, 15, 17, and 18) and only modulate the host immune system to establish long-term
one (SPI-14) for S. Typhimurium [20]. SPI-1 harbors the genes for asymptomatic infection [45].
T3SS1, which is crucial for the invasion of non-phagocytic cells Bacterial outer membrane vesicles (OMV) have been recently
such as M cells in the gut lumen and activation of pro- identified as another method used by Salmonella to transfer its

Figure 3. Salmonella and its first encounter with the host. (a) The intracellular life of Salmonella. Invasion of phagocytic and non-phagocytic
cells. Salmonella is a facultative intracellular pathogen that can be found in a variety of phagocytic and non-phagocytic cells, in which it is able to
survive and replicate. To establish this intracellular niche, the T3SS1 and -2 play a predominant role; key virulence factors are involved in accessing
and utilizing these cells [36]. After ingestion, intestinal colonization follows and Salmonella enters enterocytes and dendritic cells in the intestinal
epithelium [36]. Subsequently, Salmonella that reach the submucosa can be internalized by resident macrophages via different mechanisms: by
phagocytosis, active invasion using the T3SS1 or T3SS1-independent invasion using fimbriae or other adhesins on the bacterial surface. (1)
Salmonella-containing-vacuole. Following internalization Salmonella remains within a modified phagosome known as the Salmonella containing
vacuole (SCV) and injects a limited number of effector proteins, such as SipA, SipC, SopB/SigD, SodC-1, SopE2, and SptP into the cytoplasm. These
effectors cause rearrangements of the actin cytoskeleton and SCV morphology among other changes. (2) Replication within the SCV. Salmonella
survives and replicates within the SCV, where it is able to avoid host antimicrobial effector mechanisms. The T3SS2 is required for systemic virulence
in the mouse and survival within macrophages. (3) Transport of Salmonella to distant sites. After penetration of the M cells, the invading
microorganisms translocate to the intestinal lymphoid follicles and the draining mesenteric lymph nodes, and some pass on to the
reticuloendothelial cells of the liver and spleen. Salmonella organisms are able to survive and multiply within the mononuclear phagocytic cells
of the lymphoid follicles, liver, and spleen [36]. (b) Host–pathogen interaction in typhoid and non-typhoid Salmonella. Simplified scheme of the first
encounter between Salmonella spp. and the immune system. Specified cells such as neutrophils, macrophages, dendritic, phagocytic, and epithelial
cells recognize specific pathogen associated molecular patterns (PAMPs) and danger-associated-molecular patterns (DAMPs), thereby eliciting an
immune response. PAMPs such as LPS, Flagella, and bacterial DNA can trigger TRL4, TRL5, and TRL9, respectively. TLR-induced activation of NF-kB is
essential for the production of pro-IL-1b, pro-IL-18, which can be negatively regulated by IRAK-M [90]. The NLRs are situated in the cytosol and can
also recognize PAMPs. However, NLRP3 is triggered by a different, yet unknown, mechanism, although DAMPs are thought to play a crucial role. TLR,
toll-like receptors; LPS, lipopolysaccharide; NF-kB, regulated nuclear factor kappa-light-chain-enhancer of activated B cells; IRAK-M, IL-1R-assiociated
kinase-M; IL, Interleukin; ASC, apoptotic speck protein containing a caspase recruitment domain; NLR, NOD-like receptors (including NLRP3 and
NLRC4); MyD88, myeloid differentiation primary response gene [88].
doi:10.1371/journal.ppat.1002933.g003

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virulence factors into the cytoplasm of the host cell [46]. For patients with systemic infections caused by other Gram-negative
example, S. Typhi uses the OMV to enclose ClyA, a pore-forming bacteria [60,62]. Patients with typhoid fever demonstrate a
cytotoxin, and subsequently release this virulence factor extra- distinct and highly reproducible signature in the peripheral blood,
cellulary [47]. Moreover, it has also been shown that OMVs can shown by micro-arrays and transcriptional profiling, that changes
stimulate responses important for the activation of DCs, priming during treatment and convalescence, returning in the majority of
Salmonella-specific T and B cells, and possess pro-inflammatory and cases to a normal profile as measured in healthy uninfected
antigenic function, which makes them therefore attractive as controls [63]. Patients who do return to a normal profile may be
vaccine candidates [48]. genetically or temporarily incapable of developing an effective
Fimbrae and flagella. Fimbriae or pili are found on the immune response and may be more susceptible to re-infection,
bacterial surface and are thought to be mainly important for relapse, or the establishment of a carrier state [63]. In this respect
biofilm formation, colonization, and initial attachment to the host it is of importance to note that antimicrobial treatment—which
cells, although little is known about their true virulence potential can lead to depletion of the gut microbiome—is associated with
[20,36]. Each Salmonella serovar harbors a unique combination of prolonged deleterious effects on intestinal Salmonella colonization
fimbrial operons. Flagella are long helical filaments attached to resistance, which can result in increased fecal shedding and
rotary motors embedded within the membrane that enable carrier status (Box 1) [69]. In contrast to NTS, which is an
Salmonella species to travel to the epithelial barrier after ingestion. important cause of morbidity and mortality in patients with an
In vitro, flagellin causes upregulation of pro-inflammatory inherited or acquired immunodeficiency syndrome such as HIV
cytokines in tissue culture models [49]. However, in vivo data infection, IL-12R deficiency, or chronic granulomatous disease,
showed that the role of flagella in virulence can be dispensable and typhoid fever has not been associated with any primary or
model-dependent [50–52]. acquired immunodeficiency or underlying disease [9,70,71]. Such
Polysaccharides and other putative virulence factors. The a difference can potentially be ascribed to a difference in
polysaccharidic capsule Vi antigen is of key importance for S. signaling through PRRs where production of interleukin-17-
Typhi virulence, but notably absent in S. Typhimurium, S. producing T cells and their associated family cytokines (IL-17,
Paratyphi A, and most other Salmonella serovars. Its presence -21, -22, -26) play an important role in the dissemination of NTS
increases infectivity of S. Typhi and disease severity, and natural but not S. Typhi [9].
infection is usually associated with the expression of Vi antigen in
isolated S. Typhi [53]. However, Vi negative mutants are still Pattern Recognition Receptors
able to cause a typhoid-like illness in human volunteers [54]. Two Salmonella spp. expresses multiple PAMPs, most notably T3SS,
widely separated chromosomal regions, ViaA together with ViaB, flagella, fimbrae, LPS (Vi antigen), and bacterial DNA, which are
located on SPI-7, are needed for Vi synthesis [55]. Recently it has recognized by PRRs (Figure 2). Not surprisingly, TLR4 plays an
been hypothesized that the Vi capsule can prevent host-pathogen important role in invasive Salmonellosis. TLR4-deficient mice
recognition by preventing lipopolysaccharide (LPS) recognition
by pattern recognition receptors (PRRs) [56]. In this way S.
Typhi does not elicit a neutrophil influx in the small bowel but is
able to disseminate systemically and lead to a persistent bacterial Box 1. Salmonella and the Gut Microbiome
infection [3]. Other well-documented virulence factors include
In recent years it has become clear that the intestinal
ion transporters and superoxide-dismutases [36].
microbiome, consisting of more bacteria than the total
number of cells in the human body, can be seen as an
The Host Defense exteriorised organ that exerts numerous functions in the
host response against Salmonellosis. The gut hosts
PRRs, most notably the Toll-like receptors (TLR) and NOD-
,161014 bacteria from 500–1,000 different species of
like receptors (NLR), are the first component of the immune
which three bacterial divisions—the Firmicutes (Gram-
system to detect host invasion by pathogens, initiate immune positive), Bacteroides (Gram-negative), and Actinobacteria
responses, and form the crucial link between innate and adaptive (Gram-positive)—dominate. The healthy gut microbiome
immunity [57,58]. PRRs recognize conserved motifs on pathogens can protect against epithelial cell injury by producing toxic
termed ‘‘pathogen-associated-molecular-patterns’’ (PAMPs) and metabolites known to repress Salmonella virulence gene
are also able to recognize endogenous danger signals or ‘‘danger- expression, optimizes host immune systems, and mediates
associated molecular-patterns’’ (DAMPs). During invasive Salmo- pathogen clearance from the gut lumen after non-
nella infection, PAMPs and DAMPs initiate the innate immune typhoidal Salmonella diarrhea [69,91]. Of importance,
system leading to activation and recruitment of neutrophils and antimicrobial treatment depletes the gut microbiome
macrophages and the production of pro-inflammatory cytokines, and is associated with prolonged deleterious effects on
most notably Interleukin (IL)-6, IL-1b, tumor necrosis factor intestinal Salmonella colonization resistance, which can
(TNF)-a, and interferon-gamma (IFN)-c (Figure 3b) [59–65]. IFN- result in increased fecal shedding and carrier status
c plays a central role in the control of persistent infection by induction [69,92,93]. Ingeniously, S. Typhimurium is able
affecting the extent of macrophage activation [64]. IL-18 is to exploit a specific microbiome-derived nutrient, named
important for IFN-c release and early host resistance to Salmonella ethanolamine, in order to acquire a significant growth
infections [66]. advantage in the lumen of inflamed intestine over other
potential pathogens [94]. Similarly, S. Typhimurium viru-
During severe bacterial infection uncontrolled activation of the
lence factors have been found to induce host-driven
innate immune response can lead to detrimental systemic production of a new electron acceptor that allows the
inflammation, intravascular coagulation, tissue injury, and even- pathogen to use respiration to compete with fermenting
tually death [67,68]. This hyper pro-inflammatory response is only gut microbes [95]. The potential of microbiota-based
seen to a limited extent in patients with typhoid fever. Coagulation therapies for curing Salmonella-infected patients has
abnormalities do not become clinically apparent and serum levels opened a whole new area of research [69].
of TNF-a and IL-1b are low compared to the levels measured in

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 Box 2. Mouse Models for S. Typhi Infection Box 3. Questions for Future Research
S. Typhi infects humans exclusively. The consequent lack of Genomics
animal models has hampered the study of host–pathogen
interactions in typhoid fever. To overcome this problem N Can bacterial genotype be linked to the clinical disease
phenotype in humans?
experimental murine S. Typhimurium infection has been
used extensively as a model for typhoid fever. The N Is there a true association between bacterial genotype
intestinal pathology and inflammatory response seen in and/or the presence of a multidrug resistant plasmid and
this model resembles the changes observed in patients disease severity? If there is an association, what is the
with typhoid fever [96]. Current murine models include mechanism?
both oral and systemic (intravenous or intraperitoneal)
inoculation with or without streptomycin pretreatment Host-response
[91]. Infection of susceptible mouse strains that carry a
mutation in the gene encoding for a metal transporter N Is there a difference between S. Typhi- and S. Paratyphi-
present on the SCV membrane named Nramp1 (Slc11a1), induced enteric fever in the host gene expression
such as CL57/BL6 or BALB/C mice, produces a disease that pathways?
resembles typhoid fever upon inoculation with S. Typhi- N What is the role of DAMPs during severe typhoid fever?
murium [20]. To study chronic and persistent infection Are these danger-associated molecular patterns causing
such as can be seen in S. Typhi carriers, strains of mice the damage that occurs in the gut, or are they mere
possessing the Nramp +/+ allele, which are consequently bystanders triggering the NLRP3 inflammasome?
resistant to the infection with S. Typhimurium, are used
[64]. Of note, no allelic association was identified in
N Is a change in gut flora the reason that typhoid patients
still have an altered immune profile nine months after
humans between the Nramp alleles and typhoid suscep- infection? Moreover, do these patients have an increased
tibility, and as S. Typhimurium causes a different disease in risk for re-infection with other invasive Salmonellae?
humans than S. Typhi, conclusions regarding typhoid fever
pathogenesis derived from animal experiments must be
interpreted carefully [97]. An ingenious mouse model for S. Treatment
Typhi was proposed by making use of immunodeficient
Rag22/2 yc2/2 mice engrafted with human fetal liver
N Can we exploit a better understanding of disease
pathogenesis to lead to new therapeutic approaches?
hematopoietic stem and progenitor cells creating human- Potential immunomodulating treatment strategies for
ized mice susceptible to S. Typhi [98]. Although these invasive Salmonellosis could target the pathogen
humanized mice were able to support S. Typhi replication directly (e.g., based on drugs targeting T3SS or flagella)
and persistent infection, it did not lead to an acute lethal or target key host response proteins (e.g., TLR4, NLRC4,
infection [98]. Most recently, another murine lethal S. Typhi or IL-1b) depending on the phase of the immune
model resembling characteristic features of human ty- response.
phoid fever was created by making use of humanized
nonobese diabetic-scid IL2rcnull mice, which are engrafted N Are steroids beneficial in severe typhoid fever as has
previously been suggested [100]? And if so, what is the
with human hematopoetic stem cells (hu-SRC-SCID mice).
mechanism behind these observations?
This model, which has already been proven to be useful for
detecting new virulence determinants, could also be useful
to study host–pathogen interactions and evaluate vaccine
candidates [99]. shown that S. Typhimurium induces caspase-1 and NLRC4-
dependent IL-1b release and cell death [78]. NLRC4 is able to
recognize bacterial flagellin that—as has been hypothesized by
have increased susceptibility to Salmonella infection, and stimula- different authors—can be injected accidentally into the cytoplasm
tion of TLR4 by LPS has an important role in the development of by the T3SS causing cytosolic perturbations [65,79–81]. However,
septic shock during S. Typhimurium infection [56,72–74]. during experimental infection mice deficient in NLRC4 are able to
Salmonella flagellin leads to TLR5 activation [49]. Intriguingly, clear S. Typhimurium just as efficiently as control mice, suggesting
the Vi capsule expressed in mutated S. Typhimurium prevents the involvement of other inflammasomes [78]. Indeed, Broz et al.
both in vitro and in vivo recognition of Salmonella LPS by TLR4 demonstrated that both NLRP3 and NLRC4 activate caspase-1 in
[50,56]. The presence of the Vi antigen on the cell surface leads to response to S. Typhimurium infection, and mice lacking both NLRP3
capsule formation, which ultimately prevents this recognition. A and NLRC4 genes show increased susceptibility to infection. In
crucial role is ascribed to the TviA regulatory protein, encoded on response to a bacterial trigger, NLRP3 and NLRC4 will recruit ASC
the S. Typhi–specific SPI-7 (viaB locus), which downregulates and caspase-1 into a single cytoplasmic focus, which subsequently
flagellin production and enhances Vi antigen attachment to the serves as the site for pro-IL-1b processing [82]. Recent work provides
cell surface [75]. The TviA protein therefore serves as a regulatory evidence that the recognition of bacterial flagellin by the NLRC4
switch affecting the ability of the host to recognize S. Typhi as an inflammasome in splenic dendritic cells triggers rapid release of IL-
intruder at crucial stages of the spread in humans. 18, which leads to IFN-c production by memory CD8+ T cells [65].
The inflammasomes, which are intracellular complexes consist- NLRC4-mediated release of IL-1b has also been shown to be flagellin
ing of caspase-1, NLRs (e.g., NLRP3- or NLRC4), and the dependent, while the bacterial trigger for NLRP3 remains unclear.
adaptor molecule ASC (apoptosis-associated speck-like protein However, NLRP3 is able to recognize SPI-2 T3SS mutants, which
containing a CARD), play a central role in the innate immune lack the capacity to replicate intracellularly; therefore, the receptor is
defense against S. Typhimurium [76]. Mice deficient in caspase-1 thought to play a role in the detection of persistent bacteria [83].
or the end product of inflammasome activation, namely IL-1b and Although these studies underscore the central role of the inflamma-
IL-18, have higher bacterial load and succumb earlier upon some during S. Typhimurium infection, further research is needed to
infection with S. Typhimurium [77]. In vitro experiments have define its role in typhoid fever [83].

PLOS Pathogens | www.plospathogens.org 6 October 2012 | Volume 8 | Issue 10 | e1002933

Apoptosis and Pyroptosis role of key virulence factors such as Vi antigen and T3SS (Box 2).
Apoptosis or programmed cell death is regarded as a protective Studies of genome differences between S. Typhi, S. Paratyphi A, S.
mechanism of host defense by preventing further release of pro- Typhimurium, and other Salmonella serovars have begun to explain
inflammatory cellular mediators [84]. Serovars of S. enterica are some of the variation in disease manifestations. The important yet
able to employ different mechanisms to induce macrophage cell undefined roles of DAMPs and NLR-recognition in typhoid fever
death [85,86]. Recently it has been proposed that activation of remain to be clarified, and these may be major players in the
caspase-1 can also trigger a form of pro-inflammatory cell death severe gut ulceration that is an important cause of the mortality.
called ‘‘pyroptosis’’ [87]. Caspase-1, which is triggered via the Despite these major advances, large gaps remain in our
flagellin-detection of the NRLC4-inflammasome, is able to cause understanding of the pathogenesis of the disease in humans.
clearance of S. Typhimurium independent of IL-1b or IL-18 by Unraveling of the pathogenesis of invasive Salmonellosis hopefully
pyroptotic macrophage death [76]. In infected cells, caspase-1- leads to new therapeutic treatment strategies, urgently needed in
induced lysis of macrophages can result in the release of bacteria the light of growing antimicrobial resistance. Box 3 summarizes
into the extracellular space, which will enable efficient reactive some important questions for future research on invasive Salmonella
oxygen species (ROS) mediated killing by neutrophils. During the pathogenesis research.
systemic phase of infection, S. Typhimurium is able to completely
suppress flagellin expression, which results in evasion of NLRC4
detection and subsequent pyroptosis [76]. Interestingly selected Search Strategy and Selection Criteria
Salmonella-infected caspase-1-deficient macrophages do not under-
Data for this review were identified by searches of PubMed,
go pyroptosis but display a form of delayed cell death with features
of autophagy [86]. with the search terms ‘‘Typhoid Fever’’ in combination with
‘‘epidemiology,’’ ‘‘clinical features,’’ ‘‘therapy,’’ and ‘‘origin.’’
‘‘Salmonella Typhi’’ or ‘‘Salmonella Typhimurium’’ in combina-
Conclusion
tion with ‘‘genome,’’ ‘‘virulence factors,’’ ‘‘toll-like receptors,’’
Significant progress has been made in our understanding of ‘‘NOD-like receptors,’’ and ‘‘inflammasome.’’ The references of
host–pathogen interactions in invasive Salmonellosis. Mouse identified articles were manually searched for further relevant
models using S. Typhimurium have been instrumental in papers, and we also searched our own reference databases. English
unraveling complex pathways and have shed new light on the and French papers were reviewed.

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