Title: Typhoidal<i>Salmonella</i>: Distinctive virulence factors and pathogenesis
Abstract: Cellular MicrobiologyVolume 20, Issue 9 e12939 MICROREVIEWFree Access Typhoidal Salmonella: Distinctive virulence factors and pathogenesis Rebecca Johnson, Rebecca Johnson orcid.org/0000-0003-1006-7586 MRC Centre for Molecular Bacteriology and Infection, Department of Life Sciences, Imperial College London, London, UKThese two authors are equal contributorsSearch for more papers by this authorElli Mylona, Elli Mylona MRC Centre for Molecular Bacteriology and Infection, Department of Life Sciences, Imperial College London, London, UKThese two authors are equal contributorsSearch for more papers by this authorGad Frankel, Corresponding Author Gad Frankel [email protected] orcid.org/0000-0002-0046-1363 MRC Centre for Molecular Bacteriology and Infection, Department of Life Sciences, Imperial College London, London, UK Correspondence Gad Frankel, MRC CMBI, Flowers Building, Imperial College London, London SW7 2AZ, UK. Email: [email protected] for more papers by this author Rebecca Johnson, Rebecca Johnson orcid.org/0000-0003-1006-7586 MRC Centre for Molecular Bacteriology and Infection, Department of Life Sciences, Imperial College London, London, UKThese two authors are equal contributorsSearch for more papers by this authorElli Mylona, Elli Mylona MRC Centre for Molecular Bacteriology and Infection, Department of Life Sciences, Imperial College London, London, UKThese two authors are equal contributorsSearch for more papers by this authorGad Frankel, Corresponding Author Gad Frankel [email protected] orcid.org/0000-0002-0046-1363 MRC Centre for Molecular Bacteriology and Infection, Department of Life Sciences, Imperial College London, London, UK Correspondence Gad Frankel, MRC CMBI, Flowers Building, Imperial College London, London SW7 2AZ, UK. Email: [email protected] for more papers by this author First published: 21 July 2018 https://doi.org/10.1111/cmi.12939Citations: 70 AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Abstract Although nontyphoidal Salmonella (NTS; including Salmonella Typhimurium) mainly cause gastroenteritis, typhoidal serovars (Salmonella Typhi and Salmonella 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 with S. Typhimurium. 1 INTRODUCTION Salmonella enterica subspecies enterica includes numerous pathogens of warm-blooded animals including humans. This subspecies is further classified into serovars, based on O (lipopolysaccharide) and H (flagellar) antigens (Brenner, Villar, Angulo, Tauxe, & Swaminathan, 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 nontyphoidal Salmonella (NTS). The most common NTS, and the most common cause of gastroenteritis, are the broad host range serovars Salmonella Enteritidis and Salmonella 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, Dougan, Kingsley, Heyderman, & Gordon, 2012). Typhoidal serovars, which cause typhoid (or enteric) fever, are Salmonella Typhi and Salmonella 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 gall bladder. Symptoms of typhoid typically develop 10–14 days post-ingestion and include fever, headache, muscle aches, stomach pain, and constipation or diarrhoea (Parry, Hien, Dougan, White, & Farrar, 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 gall bladder (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, Smith, & Maire, 2013). 2 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 haplotype 58 (H58). In many areas of South and Southeast Asia and sub-Saharan Africa, H58 is the dominant S. Typhi haplotype, and its introduction and spread within a region are 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 multidrug 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, has 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. 3 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, Xavier, Santos, & Bäumler, 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, Tiffany, Bronner, & Bäumler, 2018), the most apparent being that in humans, S. Typhimurium predominantly causes gastroenteritis rather than systemic disease. At the genomic level, although 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, Forest, Lepage, Leclerc, & Daigle, 2010). Additionally, both S. Typhi 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., 2016; Elhadad, McClelland, Rahav, & Gal-Mor, 2015; 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., 2018; Raffatellu, Chessa, et al., 2005; Song, Gao, & Galán, 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. 4 S. Typhi-SPECIFIC VIRULENCE FACTORS 4.1 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, Waxin, Ecobichon, & Popoff, 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, Chessa, et al., 2005). TviA is a positive regulator promoting expression of the viaB locus (Virlogeux et al., 1995), whereas 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, whereas it is upregulated in tissues during systemic dissemination, where it prevents antibody-mediated induction of neutrophil responses (Hiyoshi, Wangdi, et al., 2018; 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; Figure 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 (Figure 1), which is one of the characteristics of S. Typhi infection that make it distinct from the S. Typhimurium infection (Raffatellu, Chessa, et al., 2005; Winter, Raffatellu, Wilson, Rüssmann, & Bäumler, 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). Figure 1Open in figure viewerPowerPoint Molecular pathogenesis of Salmonella Typhi. S. Typhi uses an array of virulence factors during infection, which are distinct from nontyphoidal Salmonella (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., t1865) are probably associated with pathogenesis. Oxygen availability, fever-like temperatures (42°C), and the presence of bile results in differential SPI-1 T3SS regulation between typhoidal and nontyphoidal Salmonella. Although the role of SPI-2 T3SS during infection with S. Typhi remains unclear, S. Typhi also lacks a number of SPI-2 T3SS effectors found in S. Typhimurium. S. Typhi expresses the Vi antigen that hinders antibody binding and complement activation, as well as recognition of lipopolysaccharide 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 with 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 with S. Typhimurium S. Paratyphi A is missing the viaB and thus does not express the Vi capsule (Bueno et al., 2004). Instead, S. Paratyphi A averts antibody binding and antibody-mediated complement activation by elaboration of long O-chains (Hiyoshi, Wangdi, et al., 2018). Importantly, S. Typhi carries a mutation in the gene responsible for formation of the long lipopolysaccharide 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, Wangdi, et al., 2018). 4.2 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, Ugalde, & Galán, 2008). The toxin is expressed exclusively when S. Typhi is intracellular and localized 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, as well as to DNase I protein families (Haghjoo & Galán, 2004), whereas 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 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 (Figure 1; Chang, Song, & Galán, 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 gall bladder cancer (Nath, Singh, & Shukla, 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. 4.3 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 (Figure 1; Hayashi et al., 2001; Kortmann, Brubaker, & Monack, 2015). Although most NTS exhibit phase variation via alternate expression of two flagellin 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, Newton, Schoolnik, & Stocker, 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). 5 DIFFERENCES IN THE T3SSs 5.1 The SPI-1 and SPI-2 type III secretion systems Common to both typhoidal and NTS are two pathogenicity-island encoded 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, Hayward, & Koronakis, 2007; Finn, Chong, Cooper, Starr, & Steele-Mortimer, 2017), the SPI-1 T3SS is mainly active when Salmonella are extracellular and permits invasion of nonphagocytic cells, whereas 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, Soncini, Solomon, & Groisman, 1996). In S. Typhi, the SPI-1 T3SS is also required for invasion of nonphagocytic 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, Ferraro, Sabbagh, & Daigle, 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, Lepage, McClelland, & Daigle, 2012). The role of SPI-2 during the intracellular lifestyle of typhoidal serovars therefore warrants further investigation. 5.2 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; although nontyphoidal 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 gall bladder at high concentrations prior to release into the intestines. Several enteric pathogens control production of virulence factors following bile exposure, including Salmonella (Begley, Gahan, & Hill, 2005). In S. Typhimurium, expression of the SPI-1 T3SS and subsequently invasion into nonphagocytic cells are 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 characterized, but the stability of the dominant SPI-1 regulator HilD is increased approximately threefold in S. Typhi in bile, but decreased over threefold in S. Typhimurium in bile (Eade et al., 2016; Johnson et al., 2018). However, it is unknown if these changes in stability are 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, Cott Chubiz, Thaprawat, & Slauch, 2018). Differences in regulation of SPI-1 in S. Typhi may relate to gall bladder 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. 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 with 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, whereas 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 nontyphoidal serovars. Fever-like temperatures of 39–42°C, such as that experienced within an infected host, reduce invasion and motility of typhoidal but not S. Typhimurium strains (Elhadad et al., 2015). This response was further characterized 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 with 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), whereas intracellular survival is important. Importantly, the mechanisms underpinning the differences in regulation of the T3SS between typhoidal and NTS have not been well characterized. An important challenge for future studies is therefore to define the regulatory circuits controlling SPI-1 and SPI-2 expression in typhoidal strains. 5.3 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, Fluhr, Birmingham, Brumell, & Miller, 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 A: SopE2 and CigR are present in S. Paratyphi A but absent in S. Typhi, whereas SteC, SifB, and SspH2 are absent in S. Paratyphi A but present in S. Typhi (Table 1). Table 1. The T3SS effector repertoire in S. Typhimurium, S. Typhi, and S. Paratyphi A T3SS Effector Genomic location S. Typhimurium LT2 S. Typhi Ty2 (CT18) S. Paratyphi A ATCC9150 Function/comment SPI-1 SopB (SigD) SPI-5 STM1091 t1828 (STY1121) SPA1759 Inositol phosphatase that activates the kinase Akt, inhibiting apoptosis of infected cells both at early and late time points during infection (Finn et al., 2017; Knodler, Finlay, & Steele-Mortimer, 2005). Contributes to Salmonella invasion (Raffatellu, Wilson, et al., 2005). SopA STM2066 t0808a (STY2275)a SPA0805a E3 ubiquitin ligase that promotes polymorphonuclear leucocyte transmigration and stimulates immune signalling via members of the TRIM E3 ligases (Kamanova, Sun, Lara-Tejero, & Galán, 2016; Zhang, Higashide, McCormick, Chen, & Zhou, 2006). Pseudogenisation in S. Typhi linked with pseudogenisaton of sopE2 (Valenzuela et al., 2015) SipA SPI-1 STM2882 t2784 (STY3005) SPA2740 Promotes actin polymerisation near adherent bacteria, contributes to Salmonella invasion (Lilic et al., 2003; Raffatellu, Wilson, et al., 2005). Persists in infected cells to regulate SCV morphology in cooperation with SifA (Brawn et al., 2007). SipB SPI-1 STM2885 t2787 (STY3008) SPA2743 SPI-1 T3SS translocon component (Myeni, Wang, & Zhou, 2013). Induces apoptotic cell death in macrophages via binding to caspase-1 (Hersh et al., 1999) SipC SPI-1 STM2884 t2786 (STY3007) SPA2742 SPI-1 T3SS translocon component (Myeni et al., 2013). Bundles and nucleates actin promoting Salmonella invasion (Hayward & Koronakis, 1999; Myeni & Zhou, 2010). SipD SPI-1 STM2883 t2785 (STY3006) SPA2741 SPI-1 T3SS translocon component. Required for effector translocation into host cells (Kaniga, Trollinger, & Galán, 1995). Differentially evolved between typhoidal and nontyphoidal serovars (Eswarappa et al., 2008) SptP (StpA) SPI-1 STM2878 t2780 (STY3001) SPA2736 GTPase-activating protein domain containing effector that antagonises the activity of SopE/SopE2 to promote host cytoskeletal recovery (Fu & Galán, 1999). Inhibits MAPK signalling (Lin, Le, & Cowen, 2003). Promotes intracellular replication at late stages of infection (Humphreys et al., 2009). Differentially evolved between typhoidal and