Early studies of Salmonella entry showed that the membrane ruffles that are generated by the pathogen morphologically resemble lamellipodia, flat sheet-like extensions of the plasma membrane that the cell uses to adhere to and move over surfaces (Takeuchi, 1967). Lamellipodia formation requires the host WAVE Regulatory Complex (WRC; Bisi et al., 2013), a heteropentameric complex composed of WAVE (WASP family verprolin homolog), Abi (abl-interactor), Cyfip (cytoplasmic FMR1 interacting protein), Nap1 (NCK-associated protein 1), and HSPC300 (heat shock protein C300), or their homologs (Chen et al., 2010). WAVE is a nucleation promoting factor (NPF), an activator of the Arp2/3 complex, the cells major actin nucleation machinery, which in turn drives the formation of branched networks of actin filaments that form the lamellipodium (Bisi et al., 2013). It is well-established that the same pathway promotes the pronounced membrane ruffling associated with Salmonella entry (Hanisch et al., 2010), and recent work has identified the multiplicity of signaling events that must be coordinated by Salmonella in order to correctly subvert WRC function (Figure 2).

The activity of the WRC is governed by a remarkable interplay between Rac1 and Arf1 GTPases (Singh et al., 2017), which are both required to recruit the WRC to the membrane and promote subsequent Arp2/3 activation (Koronakis et al., 2011). As described above, the Salmonella entry effector SopE is a GEF mimic which directly activates Rac1, a process that possibly requires the host membrane protein caveolin (Lim et al., 2014). However, Salmonella does not encode any known Arf GEFs, and instead must exploit host regulatory networks to achieve the Arf1 activation necessary for WRC activation (Humphreys et al., 2012). Arf1 is best known for its activities in membrane trafficking at the Golgi membrane, but can be recruited to the plasma membrane by the cellular GEF ARNO (Arf nucleotide-binding-site opener). ARNO is maintained in the cytosol in an autoinhibited conformation but is recruited and activated at the plasma membrane via GTP-bound Arf6 and acidic phospholipids such as PI(3,4,5)P₃ that interact with the protein's Pleckstrin Homology (PH) domain (Cohen et al., 2007). The production of PI(3,4,5)P₃ at sites of Salmonella internalization is triggered by SopB, but Arf6 is activated by the cellular GEFs BRAG and EFA6 (Humphreys et al., 2013). These host factors are known to be activated at least in part through interaction with PI(4,5)P₂, though the means by which they are specifically recruited and activated by the pathogen has yet to be uncovered.

GTPases such as Rac1 and Arf1 are turned off by proteins called GAPs, GTPase activating proteins, which trigger hydrolysis of bound GTP to GDP. Salmonella encodes its own Rac1 GAP, SptP, which following pathogen uptake, returns the cell's signaling to the resting state (Fu and Galan, 1999). As is the case for the GEFs, Salmonella encodes no known Arf GAP, but instead hijacks host proteins (Davidson et al., 2015). Remarkably however, the function of these extends beyond simply turning off signaling. When the expression of Arf6 GAPs ACAP1 and ADAP1 or the Arf1 GAP ASAP1 was knocked down by RNAi, entry of Salmonella into cultured cells was significantly impaired (Davidson et al., 2015). This suggests that WRC-driven actin assembly requires cycles of Arf activation and inactivation. Salmonella-induced membrane ruffling thus represents a fascinating interplay between Arf and Rac1 GTPases, between host and pathogen regulatory proteins, and between positive and negative control mechanisms.

Salmonella-induced membrane ruffling has long been thought of as the driving force behind internalization, but in recent years it has become apparent that the two activities can be separable, at least in certain cell types. In cultured fibroblasts, if components of the WRC are depleted by RNAi, membrane ruffles are completely abolished but pathogen uptake can still be observed (Hanisch et al., 2010). This residual entry could be further reduced by additional Arp2/3 knockdown, suggesting that an NPF other than WAVE must be responsible for this activity. Surprisingly this NPF is not NWASP, which is activated by Cdc42 and was previously thought to be required for Salmonella invasion. In fact, an NWASP knockout cell line showed increased levels of pathogen entry (Hanisch et al., 2010), A screen of other known NPFs identified a role for the WASH complex (Hanisch et al., 2010), a heteropentamer with a similar architecture to the WRC, composed of the NPF WASH (Wiskott-aldrich syndrome protein and scar homolog), Strumpelin, SWIP (Strumpelin and WASH interacting protein), FAM21 (Family number 21, also known as Vaccina Penetration Factor or VPEF), and CCDC53 (coiled-coil domain containing protein 53).

The WASH complex was only discovered fairly recently (Linardopoulou et al., 2007; Derivery et al., 2009; Gomez and Billadeau, 2009; Jia et al., 2010) and little is known about its regulation in the cell. Its main function seems to be in promoting Arp2/3-dependent actin polymerization on endosomes and lysosomes. The WASH complex was however shown to accumulate at sites of Salmonella entry into fibroblasts, and knockdown of complex components resulted in a small, though statistically significant, impairment of pathogen uptake (Hanisch et al., 2010). The WASH complex accumulates on phagosomes and macropinosomes in Dictyostelium, where it plays a key role in recycling certain proteins to the plasma membrane and avoiding their degradation (Buckley et al., 2016). It remains to be determined whether WASH has a similar function during Salmonella uptake, or whether it plays a more direct role in actually driving actin-dependent pathogen entry. The Salmonella effectors responsible for subverting WASH have yet to be identified. It has been reported that WASH acts downstream of Rho1 (Liu et al., 2009), the Drosophila ortholog of mammalian RhoA. The Salmonella effector SopB can indirectly trigger RhoA activation (Figure 3; see below), but any link between SopB and WASH has not been reported.

While Arp2/3 is the cell's best-characterized nucleator of actin polymerization, other pathways do exist. When expression of Arp2/3 is knocked down in cultured fibroblasts, Salmonella invasion is severely reduced but is not completely abolished (Hanisch et al., 2010). This suggests the existence of Arp2/3 independent entry pathways. It was noticed that Salmonella infection of fibroblasts often led to the generation of actin stress fibers in the cells, which were specifically decorated with myosins IIA and IIB (Hanisch et al., 2011). Myosins are molecular motors and ATP hydrolysis by myosins can cause them to slide along and generate contractive forces on anchored actin filaments. When cells were treated with chemical inhibitors of mysoin II, pathogen uptake was reduced by around 50% (Hanisch et al., 2011). Importantly, this inhibition was additive with Arp2/3 RNAi, confirming that the two represent separate pathways of bacterial entry.

Entry of ΔsopB Salmonella (assumed to be driven in the main by SopE/E2) was unaffected by myosin inhibition, whereas ΔsopE/E2 strains (in which it is assumed that SopB is responsible for uptake) were efficiently inhibited, suggesting that SopB is the effector responsible for hijacking myosin II (Hanisch et al., 2011). The GTPase RhoA and its downstream target Rho Kinase are known activators of myosin II, and indeed chemical inhibition of either gave a similar phenotype to myosin inhibition for SopB-mediated uptake (Hanisch et al., 2011). Consequently a pathway can be drawn in which SopB activates RhoA, this activates Rho Kinase which in turn phosphorylates and activates myosin II-dependent contractility, allowing it to contribute to Salmonella uptake (Figure 3). The molecular detail of how the lipid phosphatase SopB activates RhoA remain to be fully determined. The products of cellular phosphatidylinositol-3-kinase (PI3K), such as PI(3,4)P₂ and PI(3,4,5)P₃, are proposed to act upstream of RhoA activation. Despite being a phosphatase that cleaves phosphate from both the 4′ and 5′ position, both PI(3,4)P₂ and PI(3,4,5)P₃ (along with PI3P) accumulate in infected cells in a SopB-dependent manner (Mallo et al., 2008), which could explain the activation of the RhoA pathway.

Formins are a family of proteins that control cell shape, adhesion, and cytokinesis by promoting actin polymerization independently of Arp2/3. The existence of Arp2/3-independent entry pathways for Salmonella made the formin family an obvious target for investigation, and indeed studies with chemical inhibitors and RNAi identified the formin FHOD1 as playing a role in pathogen uptake (Truong et al., 2013). When FHOD1 is knocked down, Salmonella ruffles are much smaller, whereas Arp2/3 knockdown leads to the production of long filopodia-like extensions rather than the typical lamellipodia-like structures. It has been suggested that FHOD1 may generate new actin filaments early in the entry process, which Arp2/3 can then bind to and generate the branched actin network required for broad ruffle formation. Consistent with this, FHOD1 is recruited to Salmonella entry sites before Arp2/3 (Truong et al., 2013).

Interestingly, as with myosin II, FHOD1 lies downstream of RhoA and Rho Kinase in signaling pathways, suggesting that SopB is likely responsible for targeting the formin (Figure 3). Consistent with this, ΔsopB Salmonella showed reduced activation of FHOD1 (Truong et al., 2013). However, a similar phenotype was observed with a ΔsopE/E2 strain (Truong et al., 2013). Early studies suggested that the GEF SopE could activate RhoA in vitro (Hardt et al., 1998a), though this is not thought to happen during Salmonella invasion (Criss and Casanova, 2003). It has been suggested that FHOD1 can also be directly activated by Rac1 (Westendorf, 2001), which can certainly be activated by SopE (Patel and Galan, 2006). Consequently, the Salmonella effectors and the precise pathway that regulates FHOD1 remains to be determined conclusively.

FHOD1 is not the only non-Arp2/3 actin nucleator implicated in Salmonella entry. A recent genome-wide siRNA screen identified a role for SPIRE1 and 2 (Andritschke et al., 2016), proteins which nucleate the assembly of straight actin filaments. However, the nature of the involvement of SPIRE remains somewhat uncertain. The two isoforms seem to act at different stages of pathogen entry, with SPIRE1 effecting the initial pathogen binding event and SPIRE2 playing more of a role in establishing a replicative niche following entry (Andritschke et al., 2016). An “effectorless” Salmonella strain complemented only with either SipA (which invades cells without triggering ruffling) or with SopE (which promotes profuse ruffles) were each impaired equivalently by SPIRE knockdown. In fact, a similar phenotype was still observed in a SPI1 T3SS mutant strain in which uptake is driven by expression of the Yersinia Invasin gene (Andritschke et al., 2016), which triggers uptake by the “zipper” mechanism mediated by interaction with α5β1 integrins. Therefore, impact of SPIRE knockdown on bacterial uptake is independent of the mechanism of entry, and it is unclear whether SPIRE actually plays a direct role in the actin assembly that drives the internalization of Salmonella, or whether it has a more indirect function.

In addition to directly nucleating actin polymerization and bundling actin filaments, a yeast two-hybrid screen identified Exo70 as a potential binding partner of the SipC C-terminal domain (Nichols and Casanova, 2010). Exo70 is part of the hetero-octameric exocyst complex, the machinery that targets vesicles to the plasma membrane prior to their fusion. The exocyst complex was shown to accumulate at sites of Salmonella invasion in cultured cells, and knockdown of exocyst components led to a modest, though significant, decrease in internalization (Nichols and Casanova, 2010). Assembly and recruitment of the exocyst complex is known to require RalA, a small GTPase (Nichols and Casanova, 2010). Importantly, and consistent with a role for this complex in invasion, Salmonella activates RalA. The effector SopE is responsible for activating RalA, with a possible contribution from SopE2, though it remains uncertain whether this is a result of direct GEF activity for RalA or, perhaps more likely, an indirect pathway involving a cellular RalA GEF (Figure 4). Consistent with this hypothesis, transfection of cultured myoblasts with constitutively-active Rac1 (i.e., the confirmed substrate for SopE) leads to activation and membrane recruitment of RalA (Nichols and Casanova, 2010).

The precise role of exocyst recruitment in Salmonella uptake remains to be confirmed. The canonical internalization pathway induces pronounced membrane ruffles to drive pathogen uptake. A long-standing question has been whether the cell's membrane is truly flexible enough to form these long protrusions without extra phospholipids being incorporated. This would be a particular problem for cells that internalize multiple bacteria, as phospholipids are removed from the membrane as SCVs form. The suggestion that Salmonella may exploit the exocyst complex to promote fusion of vesicles at sites of internalization would provide a source of extra phospholipids. Interestingly, when expression of exocyst components is knocked down, Salmonella induces much smaller membrane ruffles (Nichols and Casanova, 2010). However, the exocyst complex has been reported to directly bind and recruit the WRC (Biondini et al., 2016), suggesting that its role could be in delivering not only lipids but also the protein responsible for membrane ruffling. The situation is further complicated by the observation that the exocyst can directly bind and activate Arp2/3 (Zuo et al., 2006), suggesting it could actually play a more direct role in the cytoskeletal rearrangements underlying pathogen uptake. The precise reason for Salmonella recruiting the exocyst complex remains to be determined. However, it is clear that in the cell, vesicle trafficking and membrane-localized cytoskeletal reorganization are intimately linked, a fact that a specialized invasive pathogen such as Salmonella must have evolved to exploit.

In support of this, the actin motor protein Myosin 1c has also been shown to be important for delivering signaling components to the plasma membrane for pathogen entry (Figure 4). Cholesterol-rich membrane microdomains termed lipid rafts are enriched in signaling proteins, and can be internalized and stored in the perinuclear region of the cell. Myosin 1c is required to recycle these lipid rafts back to the plasma membrane. When this pathway is blocked by Myosin 1c RNAi, Salmonella entry is reduced (Brandstaetter et al., 2012). The role of specific effectors in subverting Myosin 1c have not been investigated, however interestingly, RalA is known to bind to Myosin1c (Chen et al., 2007), suggesting a synergy with the exocyst complex. It is thus possible that Myosin 1c is required for the delivery of raft-containing vesicles to the plasma membrane, where the exocyst complex drives fusion between these vesicles and the membrane.

IQGAP1 is a protein which consists of multiple domains, including several calmodulin-binding IQ domains and a domain with homology to GTPase-activating proteins (GAPs, inactivators of Rho GTPases), which give the protein its name (Watanabe et al., 2015). IQGAP1 has been shown to localize to Salmonella entry sites, probably via its actin binding domain, and its knockdown or knockout reduces invasion significantly (Brown et al., 2007). The putative GAP domain is actually a Rho GTPase binding domain which maintains these GTPases in an active, GTP-bound state, and in its absence active Rac1 and Cdc42 no longer accumulate in infected cells (Brown et al., 2007), suggesting that following activation by SopE/E2, IQGAP1 is required to protect GTPases from inactivation.

In addition to this activity, IQGAP1 has also been proposed to act as a molecular scaffold, and binds directly to various proteins including MEK (MAPK/ERK kinase). Salmonella invasion of IQGAP1 knockout cells complemented with a Rac1/Cdc42-binding, or MEK-binding mutant of IQGAP1 was partially restored in both cases (Kim et al., 2011). In addition, entry into knockout cells complemented with the Rac1/Cdc42-binding mutant could be completely abrogated by a MEK inhibitor (Kim et al., 2011). This all suggests that the MEK actually plays an important role in the entry process. Interestingly, one of the substrates of MEK is ERK1/2 (extracellular signal-regulated kinase 1 and 2), which itself has been shown to phosphorylate Exo70 and promote assembly of the exocyst complex (Ren and Guo, 2012). In addition, IQGAP1 has been proposed to directly bind and regulate the function of the exocyst complex (Sakurai-Yageta et al., 2008). It is tempting to speculate that IQGAP1 contributes to Salmonella invasion by subverting membrane traffic in this way (Figure 4), though this has not been tested experimentally.

It is worth noting that IQGAP1 can also act as a scaffold for various phosphoinositide kinases (Choi et al., 2016). Such complexes can contain phosphatidylinositol 4-kinase III α (PI4Kα), phosphatidylinositol phosphate kinase I α (PIPKIα) and phosphatidylinositol 3-kinase (PI3K), i.e., all of the enzymes required to sequentially phosphorylate phosphatidylinositol for de novo generation of PI(3,4,5)P₃ (Choi et al., 2016). IQGAP1 could thus collaborate with SopB in the generation of PI(3,4,5)P₃ at Salmonella entry foci.

Annexins are calcium-dependent membrane binding proteins. They play a key role in membrane organization and trafficking. A number of individual annexins, such as annexins A1, A2, and A5, have been demonstrated to also bind actin, and hence function to regulate cytoskeleton-membrane dynamics. As described in the previous section, the interface between the actin cytoskeleton and the plasma membrane is of central importance to Salmonella's forced internalization and is consequently a key target for injected effectors. Perhaps predictably then, annexin A2 (AnxA2) has been reported to be required for efficient Salmonella invasion (Jolly et al., 2014).

At the plasma membrane, AnxA2 primarily forms a heterotetramer comprised of two molecules of AnxA2 and two molecules of a protein called p11. Microscopy studies showed that both AnxA2 and p11 are specifically enriched at Salmonella entry ruffles in cultured cells, and RNAi-mediated knockdown of either protein significantly reduced invasion (Jolly et al., 2014). The AnxA2/p11 complex binds directly to AHNAK, a very large phosphoprotein that is thought to cooperate in regulating membrane and cytoskeletal rearrangements. AHNAK too was recruited by Salmonella, and its knockdown reduced uptake (Jolly et al., 2014). The effectors responsible for subverting the AnxA2/p11/AHNAK pathway remain to be conclusively demonstrated. Deletion of either SopB or SopE/E2 reduced the enrichment of these proteins at entry foci, so it may be a multifactorial process, but no direct interactions have yet been reported. AHNAK is a substrate for the kinase AKT, which is known to be activated in cells by SopB. However, AKT was not required for AHNAK recruitment by Salmonella (Jolly et al., 2014).

As described above, Myosin motor proteins use the energy derived from ATP hydrolysis to move cargo along actin tracks. Myosin VI is an unusual member of the Myosin family, as unlike almost all other myosins it moves toward the minus end of actin filaments (Buss et al., 2004). Myosin VI can deliver cargo to the cell surface and plays a key role in numerous cellular functions including endocytosis. A link between Myosin VI and Salmonella uptake was first identified from in vitro reconstitution of SopE signaling at model lipid membranes (Brooks et al., 2017). SopE was shown to specifically recruit Myosin VI (along with other proteins) to these lipid bilayers. SopE activates the GTPase Rac1 to trigger WRC-dependent actin polymerization (Humphreys et al., 2012), and this assembly of actin filaments was required for Myosin VI recruitment (Brooks et al., 2017). Rac1 also has other downstream signaling targets in the cell, one of which is p21 activated Kinase (PAK). Interestingly, Rac1-induced phosphorylation of Myosin VI by PAK was also necessary, showing that two distinct pathways converge to recruit Myosin VI to the membrane (Brooks et al., 2017).

Myosin VI is important for Salmonella uptake as its knockdown reduced invasion efficiency, and its role seems to revolve around the generation of the correct phospholipid signaling platform at the site of pathogen attachment (Figure 5). As described above, SopB is responsible for generating an enrichment of PI3P, PI(3,4)P₂, and PI(3,4,5)P₃, and this activity also requires a cellular PI3K (Mallo et al., 2008), Myosin VI is required for the recruitment of PI3K to the membrane, which allows SopB to generate this lipid platform, which presumably recruits a plethora of signaling protein required for membrane ruffling and pathogen uptake (Brooks et al., 2017). One such protein that has been identified is Frabin (FGD4; FYVE, RhoGEF, and PH domain-containing protein 4), a PI3P -binding, and actin-binding, GEF for Cdc42 (Ono et al., 2000). Frabin was enriched at Salmonella entry ruffles, and its knockdown reduced invasion by around 40% (Brooks et al., 2017). It remains to be seen which other proteins are recruited by Salmonella via the Myosin VI pathway. For example Sorting Nexin 9 (Snx9) has been reported to be recruited by SopB-generated PI(3,4)P₂, and Snx9 knockdown significantly reduced entry into cultured cells (Piscatelli et al., 2016), though the role of Myosin VI upstream of Snx9 recruitment has not been investigated.

Simple cell culture models using non-polarized cells such as HeLa have been invaluable in identifying pathways exploited by Salmonella, but they may inevitably misrepresent what happens in the polarized cells of the intestinal epithelium. An example of a protein required specifically for apical entry into polarized cells is villin, and actin-binding and -severing protein that is only expressed and correctly localized in differentiated intestinal epithelial cells. Knockdown of villin leads to aberrant membrane ruffle morphology, characterized by greater ruffle diameters, and consequently causes a reduction in pathogen uptake (Lhocine et al., 2015). It is perhaps surprising that an actin severing protein is required for the correct generation of actin-driven membrane protrusions, but it has been suggested that the severing of pre-existing filaments generates an increased supply of barbed ends necessary for the new filament growth needed by Salmonella.

Logically, the activities of villin must be tightly controlled to prevent the severing of the new filaments induced by the invading pathogen, and in fact two Salmonella effectors have been shown to perform such a role (Lhocine et al., 2015). SipA binds directly to actin filaments, and when bound can prevent the severing activity of villin. In addition, phosphorylation has been reported to be required for villin's activity. A rapid increase in villin phosphorylation is seen upon Salmonella infection, however this is quickly reversed by the effector SptP, a tyrosine phosphatase. Thus, the activity of villin is highly regulated during Salmonella invasion, both spatially and temporally, to allow the highly dynamic rearrangement of the actin cytoskeleton required for correct membrane ruffle generation. Importantly, the role of villin has been confirmed in a mouse model, where villin^−/− mice, or mice expressing only a derivative of villin that lacks severing activity, are more resistant to infection by Salmonella (Lhocine et al., 2015).

Rck is an outer membrane protein encoded by the large virulence plasmid of most strains of S. enteritidis and S. typhimurium, though the rck gene is absent from other serotypes, e.g., S. choleraesuis and S. gallinarum (Heffernan et al., 1992). By using both a non-invasive E. coli strain overexpressing Rck and latex beads coated with recombinant Rck, it was demonstrated that Rck alone is able to induce entry by a receptor-mediated process. This mechanism promotes local actin remodeling, and weak, closely adherent membrane extensions, morphologically reminiscent of the “zipper mechanism” used by Yersinia species to invade non-phagocytic cells (Rosselin et al., 2010). Rck is poorly expressed in Salmonella sp. and deletion of the rck gene has no noticeable effect on the invasion of either wildtype or SPI1 mutants (Rosselin et al., 2010). The qurorm sensing molecule N-acyl homoserine lactone promotes expression of rck and results in the enhanced uptake of the bacteria (Rosselin et al., 2010). Whether rck has any effect on invasion in vivo remains unclear.

The minimal region of Rck necessary for uptake has been identified as residues 113–159. Recently it has been shown that when added to cells, this fragment can be co-immunoprecipitated with the Epidermal Growth Factor Receptor (EGFR), suggesting this is the receptor for Rck (Wiedemann et al., 2016). Consistent with this, over-expression of EGFR in cells enhanced Rck-mediated uptake, whereas either RNAi knockdown or chemical inhibition of EGFR reduced it. EGFR is usually only localized on the basolateral side of intestinal epithelial cells, suggesting that the Rck pathway may be important only after Salmonella has crossed the epithelium (Wiedemann et al., 2016). The signaling downstream of Rck activation of EGFR is fairly well-characterized (Mijouin et al., 2012; Wiedemann et al., 2012). Binding triggers autophosphorylation of certain EGFR residues, which in turn allows recruitment of cellular kinases such as c-Src, which phosphorylate further EGFR residues. This then allows recruitment of class I PI3K, which leads to activation of both Rac1 and the kinase Akt. Both Akt and Rac1 have been shown to be necessary for the Rck-induced actin rearrangements that drive uptake, via activation of Arp2/3. RNAi knockdown, dominant-negative expression and chemical inhibitors have shown that all of these components are specifically required for Rck-mediated uptake. While none of the other components of this Rck signaling cascade are involved in SPI1-promoted invasion, Rac1 is central to both pathways. As described in previous sections, Rac1 promotes SPI1-dependent membrane ruffles by activating the WRC, in conjunction with Arf1 (Humphreys et al., 2012). It remains to be seen if a similar network lies downstream of Rck.

In addition to Rck, a second outer membrane protein, PagN, has also been identified as being involved in S. typhimurium invasion (Lambert and Smith, 2008). The pagN gene is much more widely conserved than Rck, and deletion of pagN in S. typhimurium leads to a 3-fold decrease in invasion of enterocytes without altering cell adhesion. At the cellular level, the PagN-mediated entry process is poorly characterized. It was shown that actin polymerization is required during invasion (Lambert and Smith, 2008) and that PagN is able to interact with extracellular heparin proteoglycans (Lambert and Smith, 2009). Interestingly, pagN is optimally expressed in intracellular conditions, i.e., following uptake. It has been postulated that this expression pattern inside cells would prime Salmonella to be invasive upon release from the infected cell, but the true role in pathogenesis remains to be demonstrated. S. typhi also encodes PagN (also known as T2544), but deletion of the pagN gene had no effect on invasion of cells (Chowdhury et al., 2015).

Evidence for the role of Rck and PagN in invasion all stems from in vitro cell culture studies, however an additional, related outer membrane protein has been implicated in cell entry of S. typhi, and its requirement for pathogenesis has been confirmed in an animal model (Chowdhury et al., 2015). T2942 is related to Rck, and is sufficient to promote uptake of laboratory E. coli. Importantly a double t2942 and SPI1 deletion strain is minimally invasive and pathogenic in a mouse model, but expression of T2942 alone can substantially restore both properties (Chowdhury et al., 2015), suggesting SPI1 is non-essential for S. typhi. Interestingly STM3031, the homolog of T2942 from S. typhimurium, was found be redundant for invasion (Chowdhury et al., 2015), consistent with the view that SPI1 plays a much more essential role in cell entry and pathogenesis of colitis than it does for systemic disease such as typhoid fever. However, as S. typhi is a strict human pathogen, it remains to be determined whether results from mouse models will hold in the true host organism.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.