Enteropathogenic and Enterohemorrhagic Escherichia coli Infections: Translocation, Translocation, Translocation

Map.

Map, or mitochondrion-associated protein, was given this name because it targets mitochondria via an N-terminal targeting sequence (94, 99). Map displays three distinct and independent functions: (i) it interferes with the cellular ability to maintain mitochondrial membrane potential, triggering the formation of misshapen mitochondria, mitochondrial swelling, and damage (94, 99); (ii) in initial stages of EPEC/EHEC infection, Map is responsible for the transient formation of filopodium-like structures at the sites of bacterial infection, a process that is dependent on the small G protein Cdc42 (98); and (iii) Map is essential for disruption of intestinal barrier function and alteration of tight junctions, and this activity is independent of mitochondrial targeting (41). The Citrobacter rodentium mouse model of EHEC and EPEC infection (116, 179) has shown that although Map is not essential for colonization and disease (47, 130), a map mutant is recovered in lower numbers than the wild-type strain and persists longer in the mouse gut (130). In a mixed infection with 50% wild-type and 50% map mutant bacteria, the latter strain was extensively outcompeted (130). Consistent with this finding, a map mutant was found among the attenuated strains in C. rodentium (132) and EHEC signature-tagged mutagenesis screens (50), suggesting that map expression is advantageous in a competitive environment.

EspF.

EspF is a proline-rich effector protein; it contains three proline-rich repeats in EPEC (126), four in EHEC (170), and five in C. rodentium (47). EspF has been shown to play a role in the disruption of the intestinal barrier function, being required for the loss of TER, for increased monolayer permeability, and for redistribution of the tight junction-associated protein occludin (127). Map and EspF are involved in disruption of the intestinal barrier function independently; however, they both require the presence of surface-located intimin to display this activity (41). A recent study has demonstrated another level of functional similarities between EspF and Map. Like Map, EspF is targeted to the host mitochondria via its N-terminal region and is involved in mitochondrion membrane permeabilization (134, 137). Moreover, it induces release of the toxic protein cytochrome c into the cytosol and cleavage of caspases 9 and 3, indicating that EspF plays a role at the beginning of the mitochondrial death pathway (134, 137). Additionally, EspF seems to play a direct role in EPEC-induced cell death, apparently via pure apoptosis (30), and at early time points postinfection it forms a complex with cytokeratin 18 and the adaptor protein 14-3-3 (zeta isoform), a complex that is dismantled at later stages (171). Consistent with this binding activity, EspF was shown to be involved in modulating the architecture of the IF network within infected cells (171). Recent studies using human intestinal in vitro organ cultures (IVOC) have shown that EspF plays a direct role in remodeling of the brush border microvilli (152). Using the C. rodentium model, two independent studies (47, 130) showed moderate attenuation in the level of colonization by an espF mutant strain, suggesting that EspF does not have a significant role in colonization. However, a recent study has shown that an espF C. rodentium mutant is avirulent (134). The reason behind the different phenotypes is, at this stage, not known.

EspG.

Recent studies have shown that EspG triggers the formation of actin stress fibers and destruction of microtubule networks underneath adherent bacteria in fibroblasts (124, 153a). EspG interacts with tubulins and stimulates microtubule destabilization in vitro (124) and colocalizes with tubulin during infection of polarized Caco-2 cells (153a). This destabilization triggers the activation of the RhoA-ROCK signaling pathway via guanine nucleotide exchange factor (GEF-H1) activity (124). EspG displays 21% identity at the amino acid level with the Shigella flexneri effector VirA, which has been shown to trigger host microtubule destabilization, leading to Rac1 stimulation and efficient bacterial internalization (182). Indeed, espG complemented a Shigella virA mutant (53). An espG mutant strain displays only slight attenuation in animals in the rabbit EPEC infection model and the C. rodentium mouse model (47, 53, 130).

EspH.

EspH localizes to the host cell membrane and modulates the host actin cytoskeleton structure, affecting filopodium and pedestal formation (168). EspH does not play a critical role in vivo, as mutant strains show only slight attenuation in the C. rodentium mouse model (47, 130). The precise role of EspH in infection is currently not known.

SepZ.

SepZ is the most recent LEE-encoded effector to be identified; its translocation has not yet been attributed to a specific phenotype or function (89).

EspB.

In addition to its role in translocation (181), EspB was reported to have an effector activity (166). Cytosolic EspB localizes to the region of bacterial attachment (166), and cells transfected with EspB display altered morphology with a reduced number of stress fibers (165). Additionaly, EHEC EspB has been shown to bind α-catenin, a cytoskeleton-associated molecule, consistent with a role in modulating the host cell cytoskeleton (105); EPEC EspB has been shown to bind alpha(1)-antitrypsin (AAT) (101). Indeed, EPEC-mediated hemolysis of RBC and actin polymerization were strongly reduced by AAT, suggesting that AAT could interfere with type III secretion by inhibiting the correct formation of the translocation pore (101).

Tir.

Tir (transmembrane intimin receptor) localizes to the host cell plasma membrane (43, 97). Containing two membrane-spanning transmembrane domains, Tir forms a hairpin-like structure with both its C and N termini located within the host cell and the region between the two transmembrane domains forming an extracellular loop, exposed on the surface of the cell, which interacts with intimin (42, 79, 95). Like intimin in the bacterial outer membrane (167), plasma membrane-bound Tir is a dimer (115). The conformation of the intimin dimer is such that each of the two Tir-binding domains interacts with Tir molecules belonging to different Tir dimers. This binding pattern generates a reticular array-like conformation that clusters Tir under adherent bacteria (167). Tir intracellular amino and carboxy termini interact with a number of focal adhesion and cytoskeletal proteins, linking the extracellular bacterium to the host cell cytoskeleton (17, 70). These interactions lead to the formation of actin-rich pedestals beneath adherent bacteria. After delivery into the host cells, EPEC Tir is phosphorylated on two serine residues (S434 and S463) by host kinases, resulting in a shift in molecular mass (175). It has been suggested that the sequential addition of two phosphate groups triggers conformational changes in Tir structure that supply the necessary energy for insertion of Tir into the plasma membrane (175), although Tir was shown to be targeted to the plasma membranes of RBCs in the absence of detectable protein phosphorylation (153). EPEC Tir is also phosphorylated on tyrosine 474 (Y474_P); this modification is crucial for its ability to promote actin polymerization following infection of epithelial cells in vitro (95). However, recent studies have shown the existence of an alternative, Y474_P-independent mechanism of EPEC Tir-induced actin polymerization in infected human intestinal organ cultures ex vivo (Y. Chong et al., unpublished results). Significantly, EHEC Tir lacks Y474 and promotes actin polymerization through another mechanism that will be discussed further in this review (16, 48, 49, 93).

Tir and Map, which share the chaperone CesMT, show antagonistic actions in regulating filopodium and pedestal formation and synergistic mechanisms to stimulate invasion, indicating that the activities of these two effectors are coordinated during infection (98).

Cif.

Cif (cycle inhibiting factor) was the first prophage-carried effector protein to be identified. It is carried on a λ prophage, which is integrated near the Bio operon and has been found in a subset of EPEC strains isolated from human and animal clinical specimens (not being present in EPEC E2348/69, EHEC O157:H7 strains, or C. rodentium). cif encodes an effector protein that acts as a bacterial cyclomodulin. It is required for the induction of an irreversible cytopathic effect, characterized by progressive recruitment of focal adhesion plaques, assembly of stress fibers, and inhibition of the cell cycle G₂/M phase transition, leading to the accumulation of inactive phosphorylated Cdk1 (121).

EspI (NleA).

EspI (also called NleA for non-LEE-encoded effector A) is carried on the prophage CP-933P (21). EspI/NleA is not required for A/E lesion formation (73, 130), and although it does not display classical Golgi apparatus-targeting motifs, once translocated it colocalizes with Golgi markers (73). A large-scale screening of clinical isolates showed that espI was present in 53% of all LEE-positive EPEC strains tested. In contrast, espI was detected in 86% of the LEE-positive EHEC strains; interestingly, there is a statistically significant association between the presence of espI and the isolation of EHEC strains from patients suffering from symptomatic infections (131). Moreover, EspI/NleA was reported to play a critical but unknown role in virulence in the C. rodentium mouse model (73, 130).

EspJ.

espJ is located in prophage CP-933U, upstream of the TccP gene (Fig. 1). It encodes a translocated effector not required for A/E lesion formation. However, mutation in espJ influenced the dynamics of clearance of the pathogen from the host's intestinal tract in both the C. rodentium and the EHEC lamb models, suggesting a role in host survival and pathogen transmission (37).

TccP/EspFu, the EHEC lost link.

TccP (Tir-cytoskeleton coupling protein) (67)/EspFu (named because it displays 24% identity at the amino acid level with EspF [15]/U-EspF [170]) is a proline-rich effector protein carried within the EHEC prophage CP-933U and translocated through the LEE-encoded FTTSS.

EPEC and EHEC translocate Tir, which links the extracellular bacterium to the cell cytoskeleton. Although both converge on neuronal Wiskott-Aldrich syndrome protein (N-WASP), the processes of Tir-mediated actin accretion by EPEC and EHEC in cultured cells differ in that EPEC Tir requires both tyrosine phosphorylation (Y474) and the presence of the host adaptor protein Nck whereas EHEC Tir lacks a Y474 equivalent and utilizes TccP/EspFu as a linker instead. Indeed, following translocation, TccP/EspFu plays an essential role in actin accretion underneath adherent EHEC, displaying an Nck-like coupling activity. TccP/EspFu associates indirectly with Tir, binds N-WASP, and stimulates Nck-independent actin polymerization (15, 67). When expressed in EPEC, TccP/EspFu restores actin polymerization activity following infection of an Nck-deficient cell line (15, 67). Purified TccP/EspFu activates N-WASP, stimulating, in the presence of Arp2/3, actin polymerization in vitro (67). Moreover, TccP/EspFu displays similar biological activity on infected human intestinal explants ex vivo (67). In addition, TccP/EspFu seems to be involved in alteration of polarized epithelial barrier function as it complements an EPEC espF mutant (170).

(i) Disruption of the normal host-commensal strain equilibrium: diarrhea.

Several factors contribute to EPEC-/EHEC-induced watery persistent diarrhea, such as loss of microvilli and malabsorption due to the brush border deficiency. Moreover, EPEC infection disrupts the barrier function through the loss of epithelial resistance and the increased monolayer permeability that occurs through disruption of the tight and adherent junctions. EPEC activates several pathways contributing to the disruption of barrier function. (i) One of these is the redistribution and activation of ezrin. Ezrin, a member of the ezrin-radixin-moesin family, is usually located in the microvilli of epithelial cells linking membrane and cytoskeleton (8, 148). During EPEC infection, ezrin is redistributed, accumulating under adherent bacteria (57, 70), and is activated by phosphorylation on threonine and tyrosine residues, leading to the disruption of intestinal epithelial tight junctions (154). (ii) Another activated pathway is the redistribution of the tight-junction proteins. During EPEC infection, occludin is dephosphorylated by host serine/threonine phosphatases and shifts from tight junctions to the cytoplasm (127, 155). Moreover, at the same time that tight-junction proteins lose their apical localization, aberrant strands appear at the lateral membrane containing claudin-1 and occludin (133). (iii) A third pathway is phosphorylation of the myosin light chain (MLC). EPEC infection enhances the association of the MLC with the cytoskeleton, and the cytoskeleton-associated MLC is phosphorylated by MLC kinase (120). MLC phosphorylation results in hydrolysis of ATP and movement of filaments past one another. This movement is associated with contraction of the ring of cytoskeletal proteins that underlies the intercellular tight junction (12, 91). Contraction of this perijunctional ring is involved in regulating tight-junction permeability (118). EPEC-induced phosphorylation of the MLC has been shown to contribute to the increase in paracellular permeability by driving contraction of the cytoskeleton (183). (iv) Finally, EPEC activates disruption of the adherent junctions. EPEC induces phosphorylation of protein kinase C, which associates with cadherins, leading to the dissociation of the cadherin/β-catenin complex, which constitutes the adherent junctions (119). EPEC/EHEC factors implicated in induction of these effects include EspF, outer membrane proteins (119), TccP/EspFu, and Map. EspF and Map play a large role in the EPEC-induced drop in TER and contribute to EPEC-driven disruption of tight junctions, as demonstrated by the redistribution of occludin during EPEC infection (41, 127). TccP/EspFu complements an EPEC espF mutant for intestinal barrier function alteration (170).

In addition to the disruption of the epithelial barrier function, EPEC/EHEC induces changes in the host cell electrolyte transport that contribute to secretory diarrhea. A decrease in cell resting membrane potential (158) and an increase in the short-circuit current (24) are observed in EPEC-infected cells, together with changes in the bicarbonate-dependent transport of chloride and stimulation of chloride secretion (80). These events are dependent on a functional TTSS. Mechanistically, EPEC induces tyrosine phosphorylation of phospholipase C-γ1, which, once activated, interacts with and cleaves phosphatidylinositol 4,5-biphosphate, releasing inositol 1,4,5-triphosphate and diacylglycerol, secondary messengers implicated in the activation of protein kinase C, which triggers a brisk secretion of ions and fluids (31, 96). Additionally, the study of the response of polarized intestinal epithelial cells to EPEC infection has recently revealed that several proteins involved in ion transport and ion channel function, such as calcium-activated chloride in the case of channel 4, are up-regulated in response to infection with a TTSS-competent EPEC strain (78).

(ii) Host cell cytoskeleton rearrangements.

In initial stages of EPEC infection (around 5 min postinfection), filopodium-like extensions are formed at the site of bacterial adhesion. These structures extend, retract, and sway from side to side for around 20 min and then are retracted into the host cell and disappear (98). This process is dependent on Map and the host GTP-binding protein, Cdc42. Down-regulation of filopodium formation is dependent on the putative GTPase-activating protein-like arginine finger motif at the C-terminal domain of Tir and is enhanced by EspH (98, 168).

Recent studies have shown a TTSS-dependent dramatic alteration in the architecture of the IF network in EPEC- and EHEC-infected epithelial cells. The IF proteins cytokeratin 18 (CK18) and cytokeratin 8 (CK8) have been shown to be recruited to the EPEC-/EHEC-induced pedestals (6); depletion of cells from CK18 diminished formation of actin-rich pedestals at the site of EPEC adhesion (6). Moreover, EPEC Tir was shown to form a complex with CK18 and the tau isoform of the adaptor protein 14-3-3 (I. F. Connerton, personal communication).

EspF was also recently implicated in subversion of the IF network (171). EspF forms, during early stages of infection, a complex with CK18 and the zeta isoform of 14-3-3; the complex disappears at a later time (171). Additionally, EspG and EspG2 are responsible for the destruction of the microtubule network located underneath adherent bacteria, which leads to the assembly of actin stress fibers by activation of the RhoA-ROCK signaling pathway via GEF-H1 (124).

The most striking effect of EPEC/EHEC infection is the massive rearrangement of the host cell actin microfilaments, triggering generation of A/E lesions. These lesions are characterized by the intimate attachment of the bacterium to the epithelial cell membrane and by the localized effacement of brush border microvilli (102). The epithelial cell beneath adherent bacteria is raised in a characteristic pedestal formation, which may extend up to 10 μm outwards from the cell to form pseudopod-like structures. Moreover, pedestals are not static: EPEC/EHEC can move across the surface of infected cells at speeds of up to 0.1 μm/s (147). EPEC-/EHEC-induced pedestals are composed of polymerized actin, IF, and other proteins normally associated with the cytoskeleton, such as focal adhesion proteins. Tir is the only known type III effector essential for A/E lesion formation by EPEC. Tir interacts via its N-terminal domain with several focal adhesion proteins including α-actinin, talin, and vinculin (19, 66, 71, 81). Focal adhesion proteins usually connect actin cytoskeleton to the membrane, either directly through interactions with the membrane phospholipids or indirectly via interaction with membrane-associated proteins. Recruitment of these proteins is not essential for pedestal formation, as deletion of the N-terminal Tir domain does not prevent A/E lesion formation (18). The C-terminal region of EPEC Tir is essential for pedestal formation. This domain includes Y474, whose phosphorylation by host kinases (95, 143, 161) is required for pedestal formation (17, 95). The C terminus of Tir recruits several host proteins to the site of bacterial attachment. However, the key event in EPEC pedestal formation is the recruitment of Nck by a 12-residue region encompassing Y474 (17, 18, 72). Nck is an adaptor protein, which in turn recruits and activates N-WASP (72). N-WASP activates the actin-nucleating Arp2/3 complex, triggering actin polymerization and pedestal formation (88, 112, 113). Additional activator proteins cortactin (20) and Gbr2 (70) are also recruited to pedestals, possibly amplifying the signal provided by Nck and N-WASP; indeed, cortactin has been shown to be required for pedestal formation (20). Other signaling and cytoskeletal host proteins that are recruited to the site of bacterial attachment include CD44 and calpactin, which are recruited independently of Tir delivery, and gelsolin, tropomyosin, and ezrin, which are recruited independently of Tir tyrosine phosphorylation. The role of these additional proteins in the pedestal is unknown (70).

EPEC and EHEC share similarities between their respective Tir molecules, the host components associated with their pedestals, and the triggered actin polymerization pathways converging on the N-WASP-Arp2/3 cascade. However, Tir-mediated actin accretion by EHEC differs in that EHEC Tir is not tyrosine phosphorylated and utilizes, instead of Nck, the bacterial effector protein TccP/EspFu (15, 17, 67, 72, 84, 95); TccP/EspFu binds N-WASP, stimulating actin polymerization and pedestal formation. The recent finding of TccP/EspFu as the second type III effector protein required for pedestal formation during EHEC infection has been an important advance in the study of bacterially driven mammalian pathways of actin signaling. The absence of a direct interaction between Tir and TccP/EspFu indicates the requirement of an additional bacterial or cellular factor(s) that will be the object of future investigation (15, 67).