Typhoidal Salmonella: distinctive virulence factors and pathogenesis

Rebecca Johnson*, Elli Mylona*, Gad Frankel@

MRC Centre for Molecular Bacteriology and Infection, Department of Life Sciences, Imperial

College London, London, United Kingdom

*These two authors are equal contributors

@
Corresponding author

Gad Frankel, MRC CMBI, Flowers Building, Imperial College, London, SW7 2AZ

g.frankel@imperial.ac.uk

Running title: Pathogenesis of Salmonella Typhi

This article has been accepted for publication and undergone full peer review but has not been
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differences between this version and the Version of Record. Please cite this article as doi:
10.1111/cmi.12939

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Summary

While non-typhoidal Salmonella (NTS) (including S. Typhimurium) mainly cause gastroenteritis,

typhoidal serovars (S. Typhi and S. Paratyphi A) cause typhoid fever, the treatment of which is

threatened by increasing drug-resistance. Our understanding of S. Typhi infection in human remains

poorly understood, likely due to the host restriction of typhoidal strains and the subsequent popularity

of the S. Typhimurium mouse typhoid model. However, translating findings with S. Typhimurium

across to S. Typhi has some limitations. Notably, S. Typhi has specific virulence factors, including

typhoid toxin and Vi antigen, involved in symptom development and immune evasion, respectively. In

addition to unique virulence factors, both typhoidal and NTS rely on two pathogenicity-island encoded

type III secretion systems (T3SS), the SPI-1 and SPI-2 T3SS, for invasion and intracellular replication.

Marked differences have been observed in terms of T3SS regulation in response to bile, oxygen and

fever-like temperatures. Moreover, approximately half of effectors found in S. Typhimurium are either

absent or pseudogenes in S. Typhi, with most of the remaining exhibiting sequence variation.

Typhoidal-specific T3SS effectors have also been described. This review discusses what is known about

the pathogenesis of typhoidal Salmonella with emphasis on unique behaviours and key differences

when compared to S. Typhimurium.

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Introduction

Salmonella enterica subspecies enterica includes numerous pathogens of warm-blooded animals

including humans. This subspecies is further classified into serovars, based on O (LPS) and H (flagellar)

antigens (Brenner et al., 2000). In humans, infection with Salmonella broadly results in two outcomes

– either self-limiting gastroenteritis, or invasive systemic typhoid fever. The disease outcome

predominantly depends on the infecting serovar, and as such, Salmonella serovars are classified into

two groups: typhoidal and non-typhoidal Salmonella (NTS). The most common NTS, and the most

common cause of gastroenteritis, are the broad host range serovars S. Enteritidis and S. Typhimurium

(CDC, 2015). However, recently NTS variants have been associated with invasive systemic disease and

high mortality rates within immunocompromised patients in sub-Saharan Africa (Feasey et al., 2012).

Typhoidal serovars, which cause typhoid (or enteric) fever, are S. Typhi and S. Paratyphi A (Dougan &

Baker, 2014). Recent studies estimate that there are approximately 10-20 million cases of typhoid per

year, resulting in 100,000-200,000 deaths (Antillón et al., 2017; Mogasale et al., 2014).

Following ingestion, S. Typhi crosses the intestinal epithelium and disseminates to systemic sites,

including the liver, spleen, bone marrow and gallbladder. Symptoms of typhoid typically develop 10-

14 days post-ingestion, and include fever, headache, muscle aches, stomach pain, and constipation or

diarrhoea (Parry et al., 2002). The symptoms of S. Paratyphi A infection are similar, but often milder

(Dobinson et al., 2017). With appropriate antibiotic treatment, typhoid has a mortality rate of

approximately 1% (Parry et al., 2002). Following recovery from acute disease, approximately 3-5% of

infected individuals will continue to shed S. Typhi for several months to years (Gunn et al., 2014).

During chronic carriage, S. Typhi, and S. Paratyphi A can persist asymptomatically within the

gallbladder (Dongol et al., 2012; Gunn et al., 2014). As typhoidal serovars are human restricted, carriers

represent a key reservoir for S. Typhi, which contribute to the transmission and dissemination of typhoid

(Pitzer et al., 2014; Saul et al., 2013).

The global spread of H58 S. Typhi and drug-resistant typhoid

A concerning aspect of S. Typhi epidemiology is the recent expansion and global spread of one

haplotype (H58). In many areas of south and southeast Asia, and sub-Saharan Africa, H58 is the

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dominant S. Typhi haplotype, and its introduction and spread within a region is associated with typhoid

outbreaks and epidemics (Baker et al., 2011; Emary et al., 2012; Feasey et al., 2015; Holt et al., 2011;

Yan et al., 2016). The reasons underlying the global success of H58 are unexplained, but H58 strains

are often multi-drug resistant (MDR) (Wong et al., 2015). Alarmingly, the emergence of extensively

drug resistant (XDR) H58 S. Typhi strains, which are resistant to chloramphenicol, ampicillin,

trimethoprim-sulfamethoxazole, fluoroquinolones and third-generation cephalosporins, have recently

been identified in Pakistan (Klemm et al., 2018). H58 strains have previously disseminated rapidly from

the Indian subcontinent to other regions, and therefore the risk of global dissemination of XDR H58

strains is of real concern (Klemm et al., 2018; Wong et al., 2015). The increase in MDR/XDR S. Typhi

isolates poses a real threat to current treatment regimens, and if further resistance develops there is a

risk of typhoid becoming untreatable.

Differences between S. Typhi and S. Typhimurium

Despite the severity of typhoid fever and the concerning rise of antibiotic-resistant S. Typhi, the

molecular pathogenesis of the typhoidal serovars is poorly understood relative to S. Typhimurium. This

is mainly due to the absence of viable in vivo models to study typhoidal strains, as both S. Typhi and S.

Paratyphi are human restricted (Tsolis et al., 2011), and due to biosafety constraints involved in working

with typhoidal serovars (HSE, 2013). In comparison, S. Typhimurium has a broad host-range and

therefore several animal models are available to study infection in vivo (Tsolis et al., 2011). Of particular

importance is the mouse typhoid model, in which susceptible mice (Nramp ), develop a systemic
-

infection somewhat similar to typhoid in humans (Tsolis et al., 2011).

Although research using S. Typhimurium has been invaluable in understanding Salmonella virulence,

there are key differences between S. Typhimurium and S. Typhi (Hiyoshi et al., 2018a), the most

apparent being that in humans, S. Typhimurium predominantly causes gastroenteritis rather than a

systemic disease. At the genomic level, while 89% of genes are shared between the two serovars, almost

500 genes are unique to S. Typhimurium (strain LT2) and over 600 genes are unique to S. Typhi (strain

CT18) (Parkhill et al., 2001). Among the S. Typhi specific genes are those encoding important virulence

factors, including typhoid toxin and the Vi antigen (Sabbagh et al., 2010). Additionally, both S. Typhi

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and S. Paratyphi have a high proportion of pseudogenes (approximately 4%) (McClelland et al., 2004;

Parkhill et al., 2001), which are associated with a host restricted lifestyle. In comparison, in S.

Typhimurium approximately 0.9% of genes are pseudogenes (McClelland et al., 2001).

Although S. Typhimurium is a commonly used and long-held model, several studies have observed

marked phenotypic differences between S. Typhimurium and typhoidal serovars (Bishop et al., 2008;

Elhadad et al., 2015, 2016; Eswarappa et al., 2008; Johnson et al., 2017, 2018), in addition to describing

important roles for typhoidal-specific virulence factors (Haghjoo & Galán, 2004; Hiyoshi et al., 2018b;

Raffatellu et al., 2005a; Song et al., 2013; Wilson et al., 2008, 2011; Winter et al., 2015; Yang et al.,

2018). This review will therefore discuss what is known about virulence and pathogenesis of S. Typhi,

and emphasise differences between typhoidal and NTS.

S. Typhi specific virulence factors

The Vi antigen

One of the main characteristics that distinguishes S. Typhi from NTS is the production of a

polysaccharide capsule named the Vi antigen. The Vi capsule inhibits phagocytosis and confers serum

resistance (Hart et al., 2016; Wilson et al., 2011), likely by shielding the O-antigen from antibodies

(Hart et al., 2016). The genes encoding the Vi capsule comprise the viaB locus within Salmonella

pathogenicity island (SPI)-7, which also encodes the type III secretion system (T3SS) effector SopE

and a type IVB pilus (Pickard et al., 2003). The viaB locus encodes genes involved in regulation (tviA),

Vi biosynthesis (tviBCDE), export and retention of the Vi on the bacterial cell surface (vexABCDE)

(Virlogeux et al., 1995). A viaB locus is present in S. Paratyphi C and S. Dublin, but is absent from S.

Paratyphi A, and most gastroenteritis-causing serovars including S. Typhimurium (Bueno et al., 2004;

Parkhill et al., 2001; Pickard et al., 2003; Raffatellu et al., 2005a).

TviA is a positive regulator promoting expression of the viaB locus (Virlogeux et al., 1995), while it

downregulates flagellar and SPI-1 genes under high osmolarity conditions (Winter et al., 2009). Vi

expression is downregulated in the intestine, where flagella and SPI-1 play a role in invasion of

epithelial cells, while it is upregulated in tissues during systemic dissemination, where it prevents

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antibody-mediated induction of neutrophil responses (Hiyoshi et al., 2018b; Wangdi et al., 2014; Winter

et al., 2009). TviA-mediated repression of flagellin expression results in limited recognition of flagellin

by NAIP, leading to reduced levels of pyroptosis and IL-1β secretion by macrophages (Winter et al.,

2015) (Fig. 1). Vi has been reported to bind cell surface prohibitin, thus dampening inflammation

through MAPK signalling and IL-8 production (Sharma & Qadri, 2004). Reduced TLR5 and TLR4-

mediated secretion of IL-8 leads to low levels of neutrophil influx (Fig. 1), which is one of the

characteristics of S. Typhi infection that make it distinct from the S. Typhimurium infection (Raffatellu

et al., 2005a; Winter et al., 2008). Strikingly, in contrast to S. Typhimurium, human neutrophils do not

respond to the presence of S. Typhi by forming pseudopods in vitro, as the Vi interferes with

complement activation and complement-dependent chemotactic neutrophil responses (Wangdi et al.,

2014).

S. Paratyphi A is missing the viaB and thus does not express the Vi capsule (Bueno et al., 2004). Instead,

S. Paratyphi A avert antibody binding and antibody-mediated complement activation by elaboration of

long O-chains (Hiyoshi et al., 2018b). Importantly, S. Typhi carries a mutation in the gene responsible

for formation of the long LPS O-chains (fepE) and this mutation is essential for Vi-mediated

inflammation suppression (Crawford et al., 2013). Thus, the ability of the two pathogens to avoid

antibody binding, complement bactericidal effects and the induced phagocyte respiratory burst,

characteristic of (para)typhoid, was acquired through convergent evolution (Hiyoshi et al., 2018b).

The typhoid toxin

Unique to S. Typhi is expression of the typhoid toxin (Haghjoo & Galán, 2004), which is encoded on

SPI-11 (Hodak & Galán, 2013; Spanò et al., 2008). The toxin is expressed exclusively when S. Typhi

is intracellular and localised within the Salmonella containing vacuole (SCV) (Haghjoo & Galán, 2004;

Spanò et al., 2008). The typhoid toxin is an atypical AB toxin, consisting of two enzymatically active

(A) subunits (CdtB and PltA) and a homopentamer of one binding (B) subunit (PltB) (Song et al., 2013).

CdtB is homologous to the A subunit of cytolethal distending toxin (CDT), as well as to DNase-I protein

families (Haghjoo & Galán, 2004), while PltA, which has ADP-ribosyl transferase activity, and PltB

share similarities with subunits of pertussis toxin (Spanò et al., 2008). CdtB induces G2/M cell cycle

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arrest through its DNase-I-like activity by damaging the host cell DNA, and inducing the DNA-damage

response (Haghjoo & Galán, 2004). Homologues of the genes encoding the typhoid toxin are found in

S. Paratyphi A and several NTS serovars, exhibiting minor sequence differences, but are absent from

S. Typhimurium and S. Enteritidis (Suez et al., 2013).

The typhoid toxin is secreted within vesicles originating from the SCV and released into the

extracellular space (Fig. 1) (Chang et al., 2016; Spanò et al., 2008). Following export, typhoid toxin is

involved in intoxication of infected and uninfected cells through autocrine and paracrine pathways

(Chang et al., 2016; Spanò et al., 2008). Typhoid toxin enters a range of target cells through PltB-

mediated binding to glycans, predominantly those terminated with N-acetylneuraminic acid (Neu5Ac)

(Song et al., 2013). Glycosylated podocalyxin-like protein 1 (PODXL) on human epithelial cells and

CD45 on immune cells, including macrophages, have been identified as typhoid toxin receptors (Song

et al., 2013). Importantly, Neu5Ac is specific to humans, highlighting the host restriction and adaptation

of S. Typhi (Deng et al., 2014). Of note, chronic typhoidal carriage has been linked with gallbladder

cancer (Nath et al., 1997). It would therefore be interesting to investigate whether typhoid toxin is

involved in causing malignancies, through its DNA-damaging activity (Del Bel Belluz et al., 2016).

The role of typhoid toxin in disease and pathogenesis remains elusive. Mice systemically administered

with purified active typhoid toxin developed symptoms including signs of lethargy, reduced circulating

neutrophil numbers, and neurological complications manifested with motor dysfunctions, although not

fever (Song et al., 2013; Yang et al., 2018). The typhoid toxin is suggested to be involved in promoting

chronic S. Typhi infection, although the underlying mechanism warrants further investigation (Song et

al., 2010). These studies suggest that typhoid toxin is responsible for symptom development and

transition from acute to chronic state during typhoid fever and could be a possible target for alleviating

those.

The S. Typhi flagella

Flagella, while contributing to virulence, are also important activators of innate immune responses via

recognition of monomeric flagellin by TLR5 and NAIP receptors (Fig. 1) (Hayashi et al., 2001;

Kortmann et al., 2015). While most NTS exhibit phase variation via alternate expression of two flagellin

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genes (fliC and fljB), most S. Typhi strains are monophasic, specifically expressing FliC of the H:d

antigen. Interestingly, some Indonesian S. Typhi strains express H:j, a variation of H:d due to an in-

frame deletion in fliC (Frankel et al., 1989), and/or are biphasic, expressing a plasmid-encoded FljB

analogue of the H:z66 antigen (Baker et al., 2007). H:j and H:z66 antigenic variants are thought to have

recently emerged during S. Typhi evolution (Baker et al., 2008), driven by immune selection in this

high incidence region (Baker et al., 2007). This additional variation seems to play a role in S. Typhi

interactions with host epithelial cells and macrophages and partly in immune evasion (Schreiber et al.,

2015).

Differences in the T3SSs

The SPI-1 and SPI-2 type III secretion systems

Common to both typhoidal and NTS are two pathogenicity-island encoding type III secretion systems

(T3SS): the SPI-1 and SPI-2 T3SS, which are essential for Salmonella virulence. Although there is

increasing evidence of overlapping and cooperative activities (Brawn et al., 2007; Finn et al., 2017), the

SPI-1 T3SS is mainly active when Salmonella are extracellular and permits invasion of non-phagocytic

cells, whilst the SPI-2 T3SS is activated following internalisation, and functions to promote the

development of the SCV (Ramos-Morales, 2012).

In S. Typhimurium, both T3SSs are essential for invasion and intracellular replication in vitro (Galan

& Curtiss III, 1989; Ochman et al., 1996). In S. Typhi, the SPI-1 T3SS is also required for invasion of

non-phagocytic cells (Bishop et al., 2008), but the importance of the SPI-2 T3SS is less clear. Disruption

of the SPI-2 T3SS did not influence the survival of S. Typhi in THP-1 and human monocyte derived

macrophages (Forest et al., 2010), however S. Typhi strains with transposon insertions in the SPI-2

components ssaQ, ssaP or ssaN were negatively selected against during competitive growth in human

macrophages (Sabbagh et al., 2012). The role of SPI-2 during the intracellular lifestyle of typhoidal

serovars therefore warrants further investigation.

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Regulation of the T3SSs

The production and activity of T3SSs are tightly regulated, as their synthesis is energetically costly, and

T3SS components can be recognised by the host immune system (Reyes Ruiz et al., 2017; Sturm et al.,

2011). Regulatory pathways governing expression of the T3SSs in Salmonella are complex, reflecting

the myriad of environmental cues to which they are exposed. During disease in humans, typhoidal and

NTS encounter different environments; whilst non-typhoidal serovars are restricted to the small

intestine, typhoidal strains disseminate systemically. Such differences in the site of infections are likely

to contribute to differences in the control of T3SS expression.

A striking difference in the regulation of the SPI-1 T3SS between S. Typhi and S. Typhimurium relates

to their response to bile. Bile is a digestive secretion involved in the digestion and absorption of fats.

Bile serves as an important environmental cue to determine location within the host gastrointestinal

tract, and is also present in the gallbladder at high concentrations prior to release into the intestines.

Several enteric pathogens control production of virulence factors following bile exposure, including

Salmonella (Begley et al., 2005). In S. Typhimurium, expression of the SPI-1 T3SS and subsequently

invasion into non-phagocytic cells is significantly repressed following growth in bile (Prouty & Gunn,

2000). In comparison, in S. Typhi, growth in bile results in significant upregulation of the SPI-1 T3SS

and its associated genes, resulting in a significant increase in epithelial cell invasion (Johnson et al.,

2018). The mechanism(s) underpinning this difference are at present poorly characterised, but the

stability of the dominant SPI-1 regulator HilD is increased approximately three-fold in S. Typhi in bile,

but decreased over three-fold in S. Typhimurium in bile (Eade et al., 2016; Johnson et al., 2018).

However it is unknown if this increase in stability is responsible for driving changes in SPI-1 expression

in bile, or rather reflects an indirect effect on HilD stability, for example via alteration of DNA binding

(as DNA-bound HilD is proposed to be more stable) (Grenz et al., 2018). Differences in regulation of

SPI-1 in S. Typhi may relate to gallbladder carriage, or reflect different infection strategies within the

small intestines where S. Typhi elicits limited inflammatory responses.

SPI-1 expression also appears to be affected differently in response to oxygen availability between

typhoidal and NTS. Comparison of SPI-1 expression between aerobic and microaerobic cultures of S.

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Typhimurium revealed expression was broadly similar between the two conditions, but aerobically

grown bacteria were more invasive (Ibarra et al., 2010). For S. Paratyphi A however, SPI-1 expression,

and epithelial cell invasion, decreases significantly following aerobic growth compared to microaerobic

growth (Elhadad et al., 2016). Although equivalent studies have not been performed with S. Typhi,

differences in SopE-mediated invasion have been observed between the two conditions; in S. Typhi,

invasion following aerobic growth is only modestly decreased by the absence of sopE, whilst following

microaerobic growth invasion is almost entirely SopE-dependent (Johnson et al., 2017). An S.

Typhimurium strain does not show these differences, with a ΔsopE strain demonstrating a 40-60%

reduction in invasion relative to WT, following growth under either condition (Johnson et al., 2017).

Oxygen-dependent regulation of SPI-1 expression and activity in typhoidal strains may represent a way

to limit SPI-1 mediated inflammatory responses at the intestinal epithelium, where the oxygen

concentration is higher than in the lumen.

Temperature has also been reported to differentially affect T3SS-activity in typhoidal and non-typhoidal

serovars. Fever-like temperatures of 39-42°C, such as that experienced within an infected host, reduces

invasion and motility of typhoidal but not S. Typhimurium strains (Elhadad et al., 2015). This response

was further characterised in S. Paratyphi A, where a marked decrease in SPI-1 expression, as determined

by RT-qPCR and Western blotting, is observed at growth at 42°C compared to growth at 37°C (Elhadad

et al., 2015). Temperature-dependent effects were also observed on SPI-2 activity; higher temperatures

(42°C) were found to increase SPI-2 expression in both S. Paratyphi A and S. Typhimurium, however

a significant increase in SPI-2 dependent intracellular replication was observed only in S. Paratyphi A

(Elhadad et al., 2015). This response may reflect adaptation to systemic disease – once fever occurs,

typhoidal strains have disseminated, and SPI-1 mediated invasion may no longer be required (as

supported by the SPI-1 T3SS being dispensable for S. Typhimurium infection following intraperitoneal

injection in the mouse typhoid model (Galan & Curtiss III, 1989)), whilst intracellular survival is

important.

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Importantly, the mechanisms underpinning the differences in regulation of the T3SS between typhoidal

and NTS have not been well characterised. An important challenge for future studies is therefore to

define the regulatory circuits controlling SPI-1 and SPI-2 expression in typhoidal strains.

The effector proteins of the T3SS

To date, over 40 SPI-1 and SPI-2 effectors have been identified in S. Typhimurium (Table 1) (Ramos-

Morales, 2012). These effectors play diverse roles during S. Typhimurium infection, including

manipulation of the host cytoskeleton, and subversion of immune signalling, intracellular trafficking

and cell survival pathways (Ramos-Morales, 2012). Extensive description of effector functions is

beyond the scope of this review, but are well described in others, and are summarised in Table 1.

Importantly however, of the 42 effectors identified in S. Typhimurium, 21 are absent or pseudogenes

within S. Typhi (Table 1). Several of the effectors that are absent in S. Typhi have previously been

described as required for the full virulence of S. Typhimurium in vivo, including SseJ (Ohlson et al.,

2005), SpvB and SpvC (Matsui et al., 2001), SopD2 (Jiang et al., 2004), and SseI and SseK2 (Lawley

et al., 2006). Interestingly there are also differences in the effector repertoire between S. Typhi and S.

Paratyphi: SopE2 and CigR are present in S. Paratyphi but absent in S. Typhi, whilst SteC, SifB and

SspH2 are absent in S. Paratyphi but present in S. Typhi (Table 1).

The smaller effector repertoire found in typhoidal strains may reflect differences between the human

restricted lifestyle of S. Typhi and S. Paratyphi, and the broad host range lifestyle of NTS. For example,

the absence of one T3SS effector – GtgE – is directly linked with S. Typhi host restriction. Unlike S.

Typhimurium, S. Typhi is unable to survive within murine macrophages (Schwan et al., 2000; Spanò

& Galán, 2012). S. Typhimurium replication is supported by GtgE, which prevents recruitment of

Rab29, Rab32 and Rab38 to the SCV, via proteolytic degradation (Spanò et al., 2011; Spanò & Galán,

2012). However, expression of GtgE or depletion of Rab32 allows S. Typhi to survive in murine

macrophages, indicating that it is the absence of GtgE-mediated Rab32 depletion that prevents S. Typhi

growth in the murine host (Spanò & Galán, 2012).

Pseudogenisation of effectors is also likely to contribute to differences in infection. Introduction of

functional S. Typhimurium effectors into S. Typhi in which they are pseudogenes often has marked

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phenotypic consequences. For example, expression of S. Typhimurium SopD2 or SopE2 in S. Typhi

reduces invasion (Trombert et al., 2011; Valenzuela et al., 2015), expression of SopA increases

production of the proinflammatory cytokines IL-8 and IL-18 (Valenzuela et al., 2015), whilst expression

of SseJ increases invasion/intracellular replication within epithelial cells (Trombert et al., 2010).

Aside from effectors which are absent or pseudogenes, additional T3SS-associated genes are reported

to be ‘differentially evolved’ between typhoidal and non-typhoidal serovars, namely SipD, SseD, SseC,

SptP, SseF and SifA (Eswarappa et al., 2008). Of the remaining effectors that are common between S.

Typhimurium and S. Typhi, the protein identity varies: only one effector has 100% identity (SsaB),

whilst the lowest sequence identity is 87% (SipD) (Sabbagh et al., 2010). For most of these effectors it

is unknown if sequence differences translate to functional differences. However, the SPI-1 encoded

effector SptP – which has 94% identity between S. Typhi and S. Typhimurium – has been functionally

characterised in both serovars (Johnson et al., 2017).

SptP has a GTPase activating (GAP) and tyrosine phosphatase domains. In S. Typhimurium SptP is

involved in recovering the cytoskeleton following SopE/SopE2-mediated invasion via inactivation of

Cdc42 and Rac1 (Fu & Galán, 1999), and also in SCV maturation (Humphreys et al., 2009). Despite

the assumption that high protein identity correlates to functional similarity, SptP of S. Typhi contains

multiple point mutations which are clustered within its chaperone binding domain, while additionally,

strains belonging to S. Typhi H58 have a loss-of-function mutation within sptP (Wong et al., 2015).

Replacing the chaperone binding domain in S. Typhimurium with that of S. Typhi results in intracellular

instability and loss of SptP function (Johnson et al., 2017). Consistently, no phenotypic consequences

are observed following deletion of sptP in S. Typhi, with regards to cytoskeletal recovery or invasion

efficiency (Johnson et al., 2017). These results suggest that SptP is non-functional in S. Typhi, which

may have wider ramifications when considering additional effectors with varying sequence identities

between S. Typhi and S. Typhimurium.

Importantly, although there is a bias towards discussing the pseudogenisation and absence of T3SS

effectors in S. Typhi, computational or experimental screens to identify novel effectors have, to our

knowledge, not been performed with S. Typhi. However, based on homology to T3SS effectors from

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other bacterial pathogens, putative S. Typhi specific effectors have been identified, including STY1076

(NleG family), STY1423 (EspN) and STY1360 (OspB) (Hannemann & Galán, 2017). Of these,

STY1076 (t1865) can be secreted in a SPI-1 and SPI-2 T3SS-dependent manner, and translocated into

host cells, although a functional role during S. Typhi infection has not yet been described (manuscript

in preparation). As half of the currently identified S. Typhimurium effector repertoire is absent in S.

Typhi, there are potentially numerous, as yet unidentified, typhoidal-specific effectors which may be

functionally important during systemic disease.

Conclusions

Combating S. Typhi infections is a priority with WHO and The Bill & Melinda Gates Foundation, which

are part of the Coalition against Typhoid. The spread of XDR S. Typhi (Klemm et al., 2018) poses a

significant risk to human health, particularly in low and middle-income countries. While there is

concerted international effort to develop a vaccine that provides lasting immunity, in the short-term,

treatment of acutely and chronically infected individuals remains a key strategy to combat and contain

S. Typhi. The development of anti-S. Typhi compounds is dependent on achieving a greater

understanding of S. Typhi biology, gene regulation and virulence factors, particularly within the bile-

rich environment of the gallbladder.

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Figure 1. Molecular pathogenesis of S. Typhi. S. Typhi uses an array of virulence factors during

infection, which are distinct from NTS. S. Typhi secretes a pool of effectors into host cells through the

SPI-1 T3SS promoting invasion. However, various effectors present in S. Typhimurium are absent or

pseudogenes in S. Typhi including SptP and GtgE. On the other hand, S. Typhi-specific effectors (e.g.

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t1865) are probably associated with S. Typhi pathogenesis. Oxygen availability, feverlike temperatures

(42°C) and the presence of bile results in differential SPI-1 T3SS regulation between typhoidal and

NTS. While the role of SPI-2 T3SS during infection with S. Typhi remains unclear, S. Typhi also lacks

a number of SPI-2 T3SS-dependent effectors found in S. Typhimurium. S. Typhi expresses the Vi

antigen which hinders antibody binding and complement activation, as well as recognition of LPS by

TLR4 and flagellin by TLR5 resulting in reduced downstream TLR-mediated signalling. Vi production

is under the control of TviA which induces expression of the Vi-encoding locus named viaB. Through

the action of TviA, which negatively regulates flagellar and SPI-1-associated genes, the presence of

cytosolic flagellin, as well as SPI-1 T3SS components, is decreased, reducing recognition by NAIP

receptors. This leads to reduced NLRC4 inflammasome and induction of pyroptosis compared to S.

Typhimurium. Another virulence factor associated with typhoidal Salmonella is the typhoid toxin,

which is expressed when S. Typhi is intracellular. It is exported in vesicles originating from the SCV

into the extracellular space, where it can then bind to Neu5Ac-terminated receptors on target cells,

inducing G2/M cell cycle arrest and/or cell death. The action of typhoid toxin results in reduced

circulating neutrophils. Dashed lines depict reduced activation of pathways compared to S.

Typhimurium.

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Table 1. The T3SS effector repertoire in S. Typhimurium, S. Typhi and S. Paratyphi
T3SS Effector Genomic S. S. Typhi S. Paratyphi A Function/Comment
location Typhimurium Ty2 (CT18) ATCC9150
LT2
SPI-1 SopB SPI-5 STM1091 t1828 SPA1759 Inositol phosphatase that activates the kinase Akt, inhibiting
(SigD) (STY1121) apoptosis of infected cells both at early and late time points during
infection (Finn et al., 2017; Knodler et al., 2005). Contributes to
Salmonella invasion (Raffatellu et al., 2005b).
SopA STM2066 t0808 P SPA0805P E3 ubiquitin ligase which promotes polymorphonuclear leukocyte
(STY2275)P transmigration and stimulates immune signalling via members of the
TRIM E3 ligases (Kamanova et al., 2016; Zhang et al., 2006).
Pseudogenisation in S. Typhi linked with pseudogenisaton of sopE2
(Valenzuela et al., 2015)
SipA SPI-1 STM2882 t2784 SPA2740 Promotes actin polymerisation near adherent bacteria, contributes to
(STY3005) Salmonella invasion (Lilic et al., 2003; Raffatellu et al., 2005b).
Persists in infected cells to regulate SCV morphology in cooperation
with SifA (Brawn et al., 2007).
SipB SPI-1 STM2885 t2787 SPA2743 SPI-1 T3SS translocon component (Myeni et al., 2013). Induces
(STY3008) apoptotic cell death in macrophages via binding to caspase-1 (Hersh
et al., 1999)
SipC SPI-1 STM2884 t2786 SPA2742 SPI-1 T3SS translocon component (Myeni et al., 2013). Bundles and
(STY3007) nucleates actin promoting Salmonella invasion (Hayward &
Koronakis, 1999; Myeni & Zhou, 2010).
SipD SPI-1 STM2883 t2785 SPA2741 SPI-1 T3SS translocon component. Required for effector
(STY3006) translocation into host cells (Kaniga et al., 1995). Differentially
evolved between typhoidal and non-typhoidal serovars (Eswarappa et
al., 2008)
SptP SPI-1 STM2878 t2780 SPA2736 GTPase-activating protein domain containing effector that
(StpA) (STY3001) antagonises the activity of SopE/SopE2 to promote host cytoskeletal
recovery (Fu & Galán, 1999). Inhibits MAPK signalling (Lin et al.,
2003). Promotes intracellular replication at late stages of infection
(Humphreys et al., 2009). Differentially evolved between typhoidal
and non-typhoidal serovars (Eswarappa et al., 2008), unstable in S.
Typhi (Johnson et al., 2017).
SopD STM2945 t2846 SPA2801 Functions cooperatively with SopB to manipulate host membrane
(STY3073) dynamics (Bakowski et al., 2007). Contributes to Salmonella invasion
(Raffatellu et al., 2005b).

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 SopE SopEɸ Absent* t4303 SPA2564 Guanine exchange factors that activate Rac1 and Cdc42 to
bacteriophage (SL1344_2674) (STY4609) manipulate the host cytoskeleton and promote invasion (Friebel et al.,
(within SPI-7 2001; Hardt et al., 1998; Wood et al., 1996). sopE2 is not a
in S. Typhi) pseudogene within Paratyphi A (McClelland et al., 2004)
SopE2 Bacteriophage STM1855 t1023 P SPA1018
remnant (STY1987)P
SPI-2 SseF SPI-2 STM1404 t1272 SPA1449 Interacts with SseG to regulate SCV positioning close to the Golgi
(STY1716) network (Deiwick et al., 2006; Kuhle & Hensel, 2002; Yu et al.,
2016). Differentially evolved between typhoidal and non-typhoidal
serovars (Eswarappa et al., 2008)
SseG SPI-2 STM1405 t1273 SPA1448 Interacts with SseF to regulate SCV positioning close to the Golgi
(STY1715) network (Deiwick et al., 2006; Kuhle & Hensel, 2002; Yu et al.,
2016).
SteD STM2139 t0718 SPA0713 Inhibits MHC class II presentation on infected cells, inhibits T cell
(STY2367) activation (Bayer-Santos et al., 2016; Niemann et al., 2011)
GogB Bacteriophage STM2584 Absent Absent E3 ubiquitin ligase that inhibits NF-κB signalling by inhibiting the
(Gifsy-1) activity of host SCF ubiquitin ligase (Pilar et al., 2012).
SseK1 STM4157 Absent Absent Related effectors that inhibit NF-κB signalling (Günster et al., 2017;
SseK2 STM2137 Absent Absent Kujat Choy et al., 2004; Yang et al., 2015).
SseK3 Bacteriophage Absent* Absent Absent
(ST64B) (SL1344_1928)
SteC STM1698 t1612 SPA1178P Kinase the phosphorylates MAP kinases to induce actin formation
(STY1353) around SCVs and regulate intracellular replication (Geddes et al.,
2005; Odendall et al., 2012; Poh et al., 2008)
PipB SPI-5 STM1088 t1830 SPA1762 Localises to SCVs and Sifs (Knodler et al., 2003).
(STY1117)
PipB2 STM2780 t2674 SPA2636 Localises to SCVs and Sifs (Knodler et al., 2003). Recruits kinesin-1
(STY2897) to SCV membranes (Henry et al., 2006). Reorganises host endosomes
and lysosomes promoting the extension of Sifs (Knodler & Steele-
Mortimer, 2005).
SifA STM1224 t1696 SPA1626 Directs recruitment of LAMP1 enriched membranes. Required for the
(STY1264) maintenance and growth of the SCV and Sifs (Beuzón et al., 2000;
Jackson et al., 2008; Ruiz-Albert et al., 2002). Differentially evolved
between typhoidal and non-typhoidal serovars (Eswarappa et al.,
2008)
SifB STM1602 t1511 SPA1266P Member of the WxxxE family of effectors. Localises to the SCV
(STY1462) (Bulgin et al., 2010; Freeman et al., 2003). Pseudogene in Paratyphi
A (McClelland et al., 2004)

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 SopD2 STM0972 t1962P SPA1826P Interacts with Rab7 to regulate endosomal trafficking and SCV
(STY0971)P membrane dynamics (D’Costa et al., 2015). Expression of S.
Typhimurium sopD2 in S. Typhi reduces invasion (Trombert et al.,
2011)
SpiC SPI-2 STM1393 t1261 SPA1460 Involved in the translocation of SPI-2 proteins. Reported to inhibit
(SsaB) (STY1727) endosomal trafficking (Freeman et al., 2002; Uchiya et al., 1999)
SseI (SrfH) Bacteriophage STM1051 Absent Absent Interacts with IQGAP1 resulting in inhibition of macrophage and
(Gifsy-2) dendritic cell migration (McLaughlin et al., 2009). Pseudogenisation
promotes systemic dissemination (Carden et al., 2017)
SseJ STM1631 t1534 P Absent Regulates SCV membrane dynamics alongside SifA (Lossi et al.,
(STY1439)P 2008; Ruiz-Albert et al., 2002). Expression of S. Typhimurium SseJ
in S. Typhi results in increased intracellular replication (Trombert et
al., 2010)
SseL STM2287 t0576 SPA0576 Deubiquitinase that inhibits autophagic events in infected cells and is
(STY2517) required for macrophage killing (Mesquita et al., 2012; Rytkönen et
al., 2007)
SspH2 Bacteriophage STM2241 t0623 Absent E3 ubiquitin ligase that interacts with SGT1 and ubiquitinates Nod1,
remnant (STY2467) modulating immune signalling (Bhavsar et al., 2013)
SpvB pSLT pSLT039 Absent Absent ADP ribosyltransferase that depolymerises actin filaments (Lesnick et
al., 2001; Matsui et al., 2001)
SpvC pSLT pSLT038 Absent Absent Phosphothreonine lyase that inhibits MAPK signalling (Matsui et al.,
2001; Mazurkiewicz et al., 2008)
CigR SPI-3 STM3762 t3756P SPA3612 Unknown function. Required for replication in bone-marrow derived
(STY4024)P macrophages (Figueira et al., 2013; Niemann et al., 2011). Frameshift
mutation not found in Paratyphi A
GtgA Bacteriophage STM1026 Absent Absent Protease closely related to PipA and GogA. Redundantly inhibits NF-
(Gifsy-2) κB signalling (Niemann et al., 2011; Sun et al., 2016)
SPI-1/ AvrA SPI-1* STM2865 Absent Absent Acetyltransferase that inhibits JNK and NF-κB signalling, and
SPI-2 dampens proapoptotic immune responses. Also stabilises intestinal
tight junctions (Jones et al., 2008; Lin et al., 2016; Wu et al., 2012)
*Absent from SPI-1 in typhoidal strains (Sabbagh et al., 2010)
SlrP STM0800 t2087 P SPA1952P E3 ubiquitin ligase which mediates ubiquitination of mammalian
(STY0833)P thioredoxin and interacts with the chaperone ERdj3, leading to cell
death (Bernal-Bayard et al., 2010; Bernal-Bayard & Ramos-Morales,
2009; Tsolis et al., 1999)
SspH1 Bacteriophage Absent* Absent Absent E3 ubiquitin ligase that interacts with the serine/threonine protein
(Gifsy-3) (STM14_1483) kinase PKN1 and inhibits NF-κB signalling (Haraga & Miller, 2003,
2006)

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 SteA STM1583 t1493 SPA1285 Binds to phosphatidylinositol 4-phosphate (PI(4)P), localises to SCV
(STY1482) membranes and to Sifs. Implicated in the control of SCV membrane
dynamics (Domingues et al., 2014, 2016; Geddes et al., 2005)
SteB STM1629 Absent Absent Unknown function (Geddes et al., 2005)
SteE Bacteriophage STM2585 Absent Absent Unknown function (Niemann et al., 2011)
(Gifsy-1)
SpvD pSLT pSLT037 Absent Absent Cysteine protease that inhibits NF-κB signalling (Grabe et al., 2016;
Niemann et al., 2011; Rolhion et al., 2016)
GtgE Bacteriophage STM1055 Absent Absent Proteolytically targets Rab29, Rab32, and Rab38, promoting
(Gifsy-2) replication inside murine macrophages. Absence in typhoidal strains
reported to mediate host restriction (Ho et al., 2002; Spanò et al.,
2011; Spanò & Galán, 2012)
Unknown StoD Absent t1865 Absent Homologue of NleG effector family of E3 ubiquitin ligases (Tobe et
(STY1076) al., 2006). Unknown function.
PipA SPI-5 STM1087 t1831 SPA1763 Closely related proteases that are also closely related to GtgA.
(STY1115) Redundantly inhibit NF-κB signalling (Sun et al., 2016).
GogA STM2614 Absent Absent
P
= pseudogene; * = absent from S. Typhimurium LT2, where possible the locus tog of another S. Typhimurium reference (SL1344 or 14028) is given; grey
shading emphasises were differences are apparent between serovars

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