Salmonella enterica
Following adhesion between Salmonella and host epithelial cell or M cell, the T3SS encoded by chromosomal Salmonella pathogenicity island 1 (SPI-1) is activated. The bacterium then injects at least 12 effectors that trigger bacterial uptake.
Samonella inner
proteins (Sip) A and C are probably the first proteins delivered through the T3SS to the host cell [
27](Fig.
1b). Within the host cell, SipC localizes to the plasma membrane, where it aids in the translocation of other
Salmonella effectors and initiates actin nucleation. The C-terminal domain nucleates actin and the N-terminal domain of SipC bundles it, anchoring the resulting actin filaments to the cell surface below the bacterium [
28]. The injected SipA acts in synergy with SipC, as SipA binds to and stabilizes the F-actin filaments, and physically blocks the action of an actin depolymerising factor (ADF/cofilin).
The
Salmonella outer
proteins (Sop) E (SopE1 and SopE2), SopB and SptP are also delivered (in a SipC-dependent way) through the T3SS translocon into the host cell, where they are targeted to the plasma membrane [
27]. SopE and SopE2 act as guanine exchange factors (GEFs) for the Rho monomeric GTPases RhoG, Rac1 and Cdc42 [
29], which are involved in the formation of filopodia extensions and lamellipodia structures. SopB (also known as SigD) also activates Cdc42, by a poorly understood mechanism, and RhoG by activating the endogenous GEF [
30]. SopB is an inositol polyphosphate phosphatase that interferes with phosphoinositide phosphate and inositol phosphate metabolism [
31]: SopB eliminates phosphatidylinositol-3,5-biphosphate (PtdIns(3,5)P
2), PtdIns(4,5)P
2 and PtdIns(3,4,5)P
3, generating PtdIns(3)P at the site of bacterial invasion; PtdIns(3)P is probably the activator of the GEF for RhoG, and is retained in the
Salmonella-containing vacuole [
32].
Activation of RhoG, Rac1 and Cdc42 by
Salmonella leads to activation of WASp, which, complexed with monomers of globular actin and Arp2/3, initiates nucleation of actin and its polymerization into actin filaments. The induced cytoskeletal rearrangements cause membrane ruffling and culminate in macropinocytosis of bacteria by the host epithelial cell [
33]. The fact that
Salmonella mutants defective in SopE, SopE2 or SopB remain invasive and only mutants lacking all three proteins display a non-invasive phenotype illustrates the redundancy of the
Salmonella infection mechanisms [
31].
Takeuchi (1967) had already observed that the epithelial cell soon recovers its normal shape following macropinocytosis of bacteria. This recovery is brought about by the effector protein SptP, which acts as a GTPase-activating factor for the RhoGTPases targeted by the SopE and SopB proteins, thereby switching off the signal [
34].
Shigella flexneri
After having formed a pore in the membrane of M or epithelial cells, the IpaB/C complex triggers the initial events in actin polymerization. The C-terminal domain of IpaC activates Cdc42, which in turn activates Rac1, thereby inducing the formation of filopodial and lamellipodial extensions [
35].
Shigella also somehow activates the tyrosine kinase Src (pp60c), which is implicated in recruitment by phosphorylation of actin-associated proteins such as cortactin [
36].
The receptor for hyaluronic acid, CD44, once activated by IpaB, recruits ezrin to the site of entry. Ezrin recruitment seems to depend on Rho, whose activation is enhanced by the IpaB-CD44 complex [
37]. Ezrin associates with F-actin and functions as a membrane-cytoskeleton linker in the filopodial structures. Together, tyrosine kinases and small GTPases reorganize the actin cytoskeleton into entry structures similar to the focal adhesions in the host cell.
VirA is a protein encoded by a virulence plasmid and secreted in an Ipa-independent manner; it interacts with and destabilizes α/β-tubulin heterodimers, probably stimulating Rac-1 activity, which thus promotes the formation of lamellipodial structures around bacterial entry foci [
38].
Two other T3SS-secreted proteins are IpgD and IpaA. IpgD is a phosphatase with homology to SopB/SigD of
Salmonella; it generates PtdIns(5)P from PtdIns(4,5)P
2 at the site of entry, and PtdIns(5)P in turn activates the PI-3 kinase/Akt pathway, thus contributing to cell survival [
39]. IpaA complexes with the focal adhesion protein vinculin, and induces vinculin capping activity at the fast growing ends of F actin, thus promoting F actin depolymerization [
40].
These events are summarized in Fig.
2a.
Yersinia enterocolitica
Activation of integrin receptors by invasin and/or YadA triggers several intracellular signals, which are very similar to the events that precede cell division: the cytoplasmic tail of β1 chain interacts with focal adhesion kinases (FAKs), Src and the Rac-1-Arp2/3 complex, which in turn trigger slight cytoskeleton rearrangements needed for bacterial uptake [
41] (Fig.
2b, lower panel). This clustering or zipper mechanism of invasion contrasts with the trigger mechanism used by
Salmonella and
Shigella.
Yersinia species also hijack host cell phosphoinositide metabolism for their uptake. Rac-1 recruits, and Arf6 activates, the type I phosphatidylinositol-4-phosphate-5-kinase (PtdIns(4)P(5)Kα), which forms PtdIns(4,5)P
2 at the entry site, where PtdIns(4,5)P
2 may regulate phagocytic cup formation by coordinating membrane traffic and controlling F-actin production [
42].
Yersinia internalized by M cells remains intracellular, bounded by a polymerized actin-coated vacuole. The bacterium survives inside the vacuole, even though it does not replicate. This vacuole is then transported from the apical to the basolateral side of the M cell, where the bacterium is expelled and exposed to the dome region of the FAE, which is densely populated by dendritic cells, macrophages and lymphocytes (Fig.
2b, upper panel). In this way,
Yersinia crosses the epithelial barrier. However,
Yersinia species have evolved a dual strategy for avoiding destruction and establishing infection: an antiphagocytic and an anti-inflammatory strategy.
When
Yersinia invasin interacts with phagocyte integrins, the extracellular adherent bacteria transfer a set of pathogenic factors, known as Yops (
Yersinia outer
proteins) through a pYv-encoded T3SS to the target cell, and this inhibits the uptake of
Yersinia by interrupting the phagocytic pathway [
43]. The T3SS apparatus is necessary for injection of the effectors (YopE, YopP, YopT, YopH, YopO and YopM) into the host cell [
44]. Three translocator proteins are known to be required for the injection: YopB, YopD, which are believed to form a pore in the host cell membrane, and LcrV (also called V-antigen), which is localized to the tip of the needle and acts as a scaffold protein for the correct insertion of the pore formers in the membrane [
45]. LcrV is also an important anti-inflammatory agent, which has been implicated in suppression of NF-κB and interferon-γ and the secretion of inhibitory interleukin IL-10 by macrophages [
46].
YopH, YopE, YopT and YopO act indirectly on the actin cytoskeleton. YopH is a tyrosine phosphatase that dephosphorylates several macrophage proteins involved in focal adhesion, so opposing the phagocytic pathway induced by the invasin-integrin interaction [
47]. Moreover, YopH rapidly blocks the neutrophil calcium signaling pathway induced by invasin-integrin binding, thereby inhibiting their degranulation [
46]. YopE has GAP activity and there is evidence that it selectively inactivates Rac but not Rho or Cdc42 [
48]. This inhibition of Rac activity ultimately leads to the arrest of membrane ruffling, otherwise an important step in engulfment. Aili and colleagues have shown that
Yersinia devoid of YopE has high levels of Yop translocation into HeLa cells. This suggests that the GAP activity of YopE is also important in the modulation of pore formation, since YopE plays a role as a feedback inhibitor of Yop translocation [
49]. YopT also has an anti-phagocytic role, since it removes the geranylgeranylated C-terminal cysteine of RhoA; once this occurs the enzyme is released from the plasma membrane and inactivated, so preventing actin reorganization [
50]. The effector protein YopO, also known as YpkA (
Yersinia protein kinase A), possesses a C-terminal actin-binding domain necessary for YopO activation, and a domain that mimics Rho and Rac guanine nucleotide dissociation inhibitors (GDI) [
51] and maintains these factors in their GDP-bound inactive states, while its N-terminal region harbors a domain with serine/threonine kinase activity that phosphorylates a critical serine residue on G
αq protein, blocking downstream calcium signaling [
52]. Apparently, both the GDI and kinase activities of YopO are involved in disruption of the host cytoskeleton.
YopM, in contrast, has no enzymatic domain; instead it acts like a scaffolding protein, regulating the activity of two host cell kinases, PRK2 and RSK1, both involved in pathways signaling cell survival and proliferation. Association of YopM with PRK2 increases its kinase activity, which in turn activates RSK1. Once activated, the YopM-kinase complex may phosphorylate a still unknown substrate [
53].
YopP, a homologue of YopJ, orchestrates an anti-inflammatory process by exerting an inhibitory effect on both the mitogen-activated protein kinase (MAPK) and NF-κB signaling pathways [
54]. YopJ has an acetyl transferase activity that modifies the activation loop of the MAPK and Iκβ kinases
2 (see Appendix), thereby preventing their phosphorylation and subsequent activation [
55]. Inhibition of the MAPK and NF-κB signaling pathways results in rapid apoptosis of the macrophages, which is important for establishing a systemic infection.
In addition to their action on cell adhesion, the surface factors YadA and Ail may be involved in serum resistance, probably by capturing the C4b-binding protein that down-regulates the classical and lectin complement pathways [
56]. Later in infection, when
Yersinia interacts with epithelial cells,
Yersinia invasin is able to stimulate NF-κB synthesis and trigger the production of pro-inflammatory cytokines, such as IL-8, via MAPK. The YadA-ECM-β
1 interaction also leads to IL-8 production, although this occurs via a different MAPK [
57].
IL-8 and other cytokines activated by
Yersinia are chemoattractants and promoters of PMNs and macrophages. These cells are recruited to infection sites, leading to disruption of the epithelial barrier, and exposing the integrins localized on the basolateral sides of enterocytes, causing bacterial dissemination [
57]. Ulceration and necrosis of the tissue subjacent to the FAE are also characteristic of
Yersinia infections. Systemic dissemination begins 24-48 h after infection and may be enhanced by bacterial phagocytosis; the bacteria travel through the bloodstream towards the spleen and liver, where they replicate in microabscesses, forming microcolonies resistant to phagocytosis.