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Infection and Immunity, May 2005, p. 2573-2585, Vol. 73, No. 5
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.5.2573-2585.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

MINIREVIEW

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

Junkal Garmendia,{dagger} Gad Frankel,* and Valérie F. Crepin{dagger}

Centre for Molecular Microbiology and Infection, Department of Biological Sciences, Imperial College London, London, United Kingdom


   INTRODUCTION
Top
 Introduction
References
 
Escherichia coli is the most abundant facultative anaerobic gram-negative bacterium of the intestinal microflora, naturally colonizing the mucous layer of the colon. A conserved core genomic structure is common to both commensal and pathogenic strains, providing the microorganisms with mechanisms required for survival under the competitive conditions in the gut, as well as the ability to spread among hosts and survive in the environment (90). In pathogenic bacteria, the core genome framework is enhanced with novel genetic islands and small clusters of genes often associated with increased virulence (74-76). These novel genes provide the bacteria with a higher level of adaptation, leading to specific tissue targeting that can open up new ecological niches and facilitate efficient dissemination to new hosts. A striking example of highly adapted enteric bacteria that have acquired, by horizontal gene transfer, superior fitness and a competitive edge within the gut is a group of extracellular intestinal pathogens that have evolved to use attaching and effacing (A/E) lesion formation as a major mechanism of tissue targeting and infection. Among these pathogenic bacteria are the diarrheal agents enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC), highly successful pathogens that have adapted to cause infections in different hosts via related, but distinct, mechanisms of transmission.

EPEC and EHEC cause acute gastroenteritis in humans (22, 135). EPEC, the first type of E. coli to be associated with human disease, is a frequent cause of infantile diarrhea in the developing world, and EHEC, an emerging zoonotic pathogen, causes a wide spectrum of illnesses ranging from mild diarrhea to severe diseases such as hemorrhagic colitis and hemolytic uremic syndrome. Hemolytic uremic syndrome is the leading cause of acute pediatric renal failure in developed countries and is associated with the production of potent Shiga toxins (135). Strains of EHEC belonging to serogoup O157 are most commonly associated with severe human diseases.

EHEC and EPEC are sophisticated pathogens that display several virulence-associated traits, some of which are also found in other bacterial pathogens (82). The mechanisms by which EPEC and EHEC intimately adhere to epithelial cells represent the most studied feature in their pathogenesis. By adhering to intestinal epithelial cells, these bacteria subvert cytoskeletal processes to produce a histopathological feature known as an A/E lesion (reviewed in reference 64), which is characterized by localized destruction of brush border microvilli and intimate attachment of the bacteria to the plasma membranes of the host epithelial cells.

The capacity to form A/E lesions is encoded mainly by the locus of enterocyte effacement (LEE) pathogenicity island (PAI) (55, 125). The fact that the LEE is inserted into diverse chromosomal loci among various EPEC and EHEC serotypes suggests that it was acquired multiple times during the evolution of these pathogens (178).

Regulation of LEE gene expression is complex, dependent on environmental conditions, quorum sensing, and several regulators, including the EPEC adherence factor plasmid-carried per (plasmid-encoded regulator) locus and the LEE-encoded Ler (LEE-encoded regulator), GrlA (global regulator of LEE activator), and GrlR (global regulator of LEE repressor) (90).

In additon to Ler, GrlR, and GrlA (47, 128), the 5' end of the main coding strand of the LEE region encodes structural components of a type III secretion system (TTSS) commonly found in pathogenic gram-negative bacteria (82). The central part of the LEE encodes the outer membrane adhesin intimin (65, 85) and the translocated intimin receptor (Tir) (43, 97), and the 3' end encodes additional TTSS structural, translocator, and effector proteins. Important recent discoveries have highlighted the fact that the LEE-encoded TTSS is being employed to translocate effector proteins that are encoded at different locations in the genome and play an essential role in infection and disease. In this review, we summarize the current state of knowledge about EPEC and EHEC infections, concentrating on the mechanisms that the EPEC and EHEC pathogens employ, while remaining extracellular, to subvert cellular functions for the benefit of the bacteria.


   THE FILAMENTOUS TTSS
 
The TTSS pathway is associated with the virulence of many gram-negative pathogens which infect humans, animals, insects, and plants. It is utilized by the pathogens to directly translocate virulence factors from the bacteria into the targeted host cells in a single step. The TTSS apparatus is a multicomponent organelle assembled from the products of approximately 20 genes (Fig. 1 and 2).



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  		FIG. 1. Genetic organization of the EPEC/EHEC LEE and EHEC prophages CP-933U, CP-933K, and CP-933P. The image was generated using coliBase (http://colibase.bham.ac.uk/) (21).

 


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  		FIG. 2. Schematic representation of the EPEC/EHEC type III secretion apparatus. The basal body of the TTSS is composed of the secretin EscC, the inner membrane proteins EscR, EscS, EscT, EscU, and EscV, and the EscJ lipoprotein, which connects the inner and outer membrane ring structures. EscF constitutes the needle structure, whereas EspA subunits polymerize to form the EspA filament. EspB and EspD form the translocation pore in the host cell plasma membrane, connecting the bacteria with the eukaryotic cell via EspA filaments. The cytoplasmic ATPase EscN provides the energy to the system by hydrolyzing ATP molecules into ADP. SepD and SepL have been represented as cytoplasmic components of the TTSS.

 
Many components are broadly conserved among both virulence and flagellar TTSSs (3, 117, 164). The two structures consist of a succession of protein rings spanning both bacterial membranes with a needle-like extension or a flagellar hook (3, 10). The type III secretion organelles, termed the needle complexes (NC), of Salmonella and Shigella species and EPEC have been visualized by electron microscopy, revealing overall similar structures and shapes (9, 40, 100, 108, 109, 123, 151, 162, 180). The assembly of the TTSS machinery is sequential. First, the membrane-bound components, which harbor a cleavable sec-dependent signal sequence, are exported through the sec pathway to form the foundation of the NC. Cytosolic components are then added to the basal structure. The second phase of NC assembly occurs in a sec-independent manner, through the previously assembled needle base. The type III export machinery is then utilized for the secretion of components constituting the more distal elements of the apparatus. Cytoplasmic ATPases, related to the catalytic subunits of bacterial F0F1 ATPase, are believed to energize the type III export machinery and promote protein secretion (4).

In EPEC and EHEC, EscC and EscV are the main components of the outer and inner membrane ring structures, respectively (69). EscC (a mature protein of 54 kDa) belongs to the secretin family of proteins, which are found in all TTSSs (e.g., YscC in Yersinia spp. [106, 144], InvG in Samonella spp. [28, 108], HrcC in Pseudomonas spp. [46], and MxiD in Shigella spp. [11, 150, 162]) and are involved in the transport of molecules across the outer membrane by formation of a large homomultimeric annular complex (82). Although EscC has a cleavable signal sequence, which suggests that the protein is secreted through the sec pathway, its correct localization seems to be dependent on both sec and type III secretion systems as it was found in the periplasm of escN and escV mutants (69). EscV (a mature protein of 72 kDa) is predicted to have seven transmembrane domains and to form the EPEC/EHEC inner membrane ring structures. The presence of a putative signal sequence indicates that the protein is likely to be directed to the inner membrane through the sec pathway, as observed for EscV homologues in other TTSSs (e.g., PrgH in Salmonella [100, 108, 109], MxiG in Shigella [11, 150, 162], and YscV in Yersinia [26]).

The EPEC/EHEC TTSS protein EscJ, which also contains a sec-dependent signal sequence, is highly conserved within TTSSs (e.g., PrgK in Salmonella [100, 160], MxiJ in Shigella [5, 150], and YscJ in Yersinia [5]). EscJ and its homologues are lipoproteins proposed to span the periplasm, serving as a bridge between the inner and outer membrane protein rings (35, 46, 150). Those proteins share sequence similarity within the domain of the flagellar protein FliF that is believed to line the central pore of the inner membrane ring and proposed to form part of the proximal rod structure (35, 169). EscJ is required for the secretion of more distal apparatus components across the outer membrane, as an escJ mutant failed to assemble functional type III machinery. The solution structure of the EPEC EscJ has recently been determined by nuclear magnetic resonance and shows that EscJ is composed of two structured domains connected by a linker, creating a molecule that can stretch up to 10 nm in length, which approximates the width of the periplasmic spaces of gram-negative bacteria (35). The covalent linkage between the two domains is required to retain EscJ biological activity (35). This suggests that EscJ could form a cylindrical structure functioning as a bridge across the periplasmic space, connecting the outer and inner membrane rings of the NC. Structural analysis of the Salmonella NC recently revealed that PrgK is associated with the inner membrane rings and that the periplasmic rod is mainly composed of PrgJ (123). As no PrgJ homologue is present in EPEC, it is suggested that PrgK/J function is combined in a single EPEC protein, EscJ. In addition, cell fractionation has shown that, unlike PrgK, EscJ is associated mainly with the outer membrane, although small amounts of EscJ were cofractionated with inner membrane markers (35).

The EPEC/EHEC TTSS needle is likely to be composed of a single protein, EscF (180); EscF homologues are PrgI in Salmonella (123, 159), YscF in Yersinia (122), and MxiH in Shigella (86). The EscF needle forms a projection channel required for TTSS-dependent protein secretion, as an escF mutation abolishes secretion of the translocator and effector proteins (151, 180). In situ, EPEC needle length appears to be tightly regulated (180). InvJ in Salmonella (146), Spa32 in Shigella (163), and YscP in Yersinia (87) spp. were all shown to control the needle lengths in their respective TTSSs. Such a regulator has yet to be identified in EPEC/EHEC TTSSs.

The unique feature of EPEC/EHEC TTSSs is the presence of a filamentous extension to the NC-associated EscF needle, called the EspA filament (40, 51, 103, 151, 180), defining a new class of filamentous TTSS (FTTSS). The EspA filament is a polymer of the translocator protein EspA, which is likely to be the sole constituent of these hollow filamentous conduits (39, 45). Polymerization of the EspA filaments is mediated by coiled-coil interactions between EspA subunits (45). EspA filaments have been shown to bind directly to the needle protein EscF (180). Daniell and collaborators have determined the three-dimensional structure of the EspA filament (39). It consists of a helical tube with an outer diameter of ca. 120 Å and a continuous hollow central channel of ca. 2.5 nm in diameter. Although the two systems differ in size, comparisons can easily be established between the EspA filaments and the flagellar filaments in terms of helical symmetry and packing of the subunits to form the filamentous structure. In a process similar to flagellar elongation, which occurs at the distal end of the filamentous structure (56), newly synthesized EspA subunits are incorporated at the tip of the growing filament (35a). Like the NC, the EspA filament has a defined length in situ. Increasing the intracellular concentration of EspA subunits results in significantly longer EspA filaments (35a), suggesting that the amount of monomeric EspA produced in bacteria is the limiting factor and is set for a defined filament length. The EspA filament has been identified as a hollow tube, suggesting that proteins are delivered from the bacteria to the host cell through this channel. Recent studies showing that the effector protein Tir is secreted from the filament tip to the extracellular medium have provided the first experimental evidence that proteins that are secreted through the FTTSS travel along the hollow EspA filament (35a). Mature EspA filaments are important adhesion factors (23, 51), establishing a transient link between the bacterium and the host cell (103) which enables effector protein translocation. Following effector protein translocation, EspA filaments and the NC are eliminated from the bacterial cell surface; this is necessary to allow intimate bacterial attachment through intimin-Tir interactions (64, 103). Elimination of surface TTSS components parallels down-regulation of LEE gene expression in EHEC attached to eukaryotic plasma membranes (36).

Effector proteins are delivered to the host cell cytoplasm from the extremity of the EspA filament through a translocation pore formed in the plasma membrane of the host cell by the translocator proteins EspB and EspD (13, 38, 83, 107, 110). In addition, EspD is required for the biogenesis of the EspA filaments (103). This suggests that EspD could have a dual role, first as a capping protein of the EspA filament and second as an anchor connecting the filament to the host cell plasma membrane (45, 59). Translocator proteins among TTSSs present low homology. However, they share similar topologies and structural properties such as hydrophobic transmembrane domains, predicted coiled-coil domains, and the capacity to homo- and/or heteromultimerize to form a functional translocation pore (38, 44, 61, 172). EspD has been shown to interact with itself (33, 38) and with EspB (83); both EspD and EspB can be purified from eukaryotic cell membranes following EPEC infection (153, 172, 181). Functionality of the translocation pore can be demonstrated by its ability to mediate hemolysis of red blood cells (RBCs) (77, 104, 153, 174). This system has shown that EspD is the major component of the translocation pore and that EspB is required to achieve full hemolytic activity (153).

An additional protein called SepL, a homologue of SsaL in Salmonella (25), has recently been reported to play a role in the formation of the translocation apparatus (138). SepL is a soluble cytoplasmic protein which interacts with SepD (33). SepD is predicted to be cytosolic and was identified as a component of the TTSS, as a sepD mutant strain is unable to secrete translocator or effector proteins or to induce A/E lesions. In contrast, a sepL mutant secretes normal levels of effector proteins. However, as it secretes markedly reduced quantities of those proteins involved in translocation (EspA, EspB, and EspD) and is deficient in EspA filament biosynthesis, it is incapable of A/E lesion formation (47, 138). SepL is the first protein reported to have a selective activity towards effector and translocator proteins of the EPEC/EHEC TTSS. These data suggest that SepL and SepD could be involved in the "switch" from secretion of translocator proteins to secretion of effector proteins through the type III machinery.


   TYPE III SECRETION CHAPERONES
 
Chaperones are essential to assure efficient secretion and translocation of many type III secreted proteins (82, 177). The absence of chaperones can be detrimental for their specific substrates, impairing their secretion and/or stability (14, 27, 176). Type III chaperones have little sequence homology, although they show structural similarities (44, 177). Type III chaperones have been divided into four classes (IA, IB, II, and III) based on sequence homology and functional properties (140).

Up to now, five type III chaperones have been described for EPEC/EHEC. CesF belongs to class IA and chaperones the effector protein EspF (54). CesT, also a member of class IA, is a bivalent chaperone required for translocation of the effector proteins Tir and Map (1, 32, 52). Accordingly, we propose to rename CesT as CesMT. CesMT has been shown to function as a homodimer to chaperone Tir (114). In addition, CesMT binds the EscN type III ATPase, suggesting a role for the chaperone in bringing Tir into physical contact with the type III secretion apparatus (68).

CesD is a member of class II, which shares sequence similarities with other type III chaperones such as Shigella IpgC and Yersinia SycD (139, 176). CesD is a bivalent chaperone, has for substrates EspD and EspB, and localizes in both the cytoplasm and the inner membranes (173). It plays an important role in the secretion of its substrates, although a CesD mutant does not completely abolish effector protein translocation or A/E lesion formation. This is likely to be related to the existence of auxiliary chaperones: CesD2 for EspD, which also localizes to the cytoplasm and the inner membrane compartments (136), and CesAB, which is likely to be the primary chaperone for EspB. In addition, CesAB chaperones EspA and is essential for the stabilization of cytoplasmic EspA and for EspA filament biogenesis (34).


   OUTCOMES OF EFFECTOR PROTEIN TRANSLOCATION
 
The hallmark of EPEC and EHEC infection is induction of A/E lesions, characterized by local effacement of the brush border microvilli and intimate attachment of the bacterium to the plasma membrane of the host cell (102). Importantly, while remaining extracellular, EPEC and EHEC subvert the host cell cytoskeletal microtubule, intermediate filament (IF), and actin microfilament networks.

Other alterations to the host epithelial cells caused by EPEC and EHEC infections include (i) disruption of intestinal barrier function, including increased tight-junction permeability in infected polarized cells (127, 133, 170) paralleled by a decrease in transepithelial resistance (TER); (ii) loss of mitochondrial membrane potential, triggering the formation of misshapen mitochondria, indicative of mitochondrial swelling and damage (98, 99); (iii) inhibition of the cell cycle G2/M phase transition (121); and (iv) induction of cellular apoptosis (29, 30, 60). The physiological consequences of the infection include (i) production of interleukin-8 and transmigration of acute inflammatory cells, primarily polymorphonuclear cells, to the site of infection (149) and (ii) diarrhea.


   INTIMIN: THE OUTER MEMBRANE EPEC AND EHEC ADHESION MOLECULE
 
Intimin is exported via the general secretory pathway to the periplasm, where it is inserted into the outer membrane by a putative autotransport mechanism (167). Intimin has two functional regions: the N-terminal region, highly conserved between different EPEC and EHEC strains, is inserted into the bacterial outer membrane, forming a ß-barrel-like structure, and mediates dimerization (167). The C-terminal 280-amino-acid sequence of intimin (Int280) is variable, defines several intimin types (2, 65), extends from the bacterium, and interacts with receptors in the host cell plasma membrane (62). Int280 comprises three globular domains: two immunoglobulin-like domains and a C-type lectin-like domain; the latter, together with the last immunoglobulin-like domain, contains the receptor-binding site (7, 92, 111, 115). Compelling evidence has recently been accumulated showing that intimin binds, in addition to Tir, a receptor encoded by the host cell (65, 111). Two potential host cell-carried intimin receptors (Hir) have so far been identified, ß1 integrin (63) and nucleolin (156, 157), although the physiological relevance of these interactions has yet to be demonstrated. Interaction of intimin with host cells stimulates production of microvilli-like processes (142), intimin types influence gut tissue tropism (58, 141, 145), and intimin-Hir interaction has been recently shown to be essential for EPEC-induced modulation of the tight junctions (41).


   LEE-ENCODED EFFECTORS
 
Tir, Map, EspF, EspG, EspH, SepZ, and EspB are known LEE-encoded effector proteins. Below we summarize the current knowledge about their functions (Fig. 1 and 3; Table 1).



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  		FIG. 3. EPEC and EHEC translocate LEE-encoded and other effector proteins into the host cell cytosol. These effectors trigger cytoskeleton rearrangements (Tir, EspH, EspF, and EspG), disruption of the epithelial barrier (EspF and Map), cytotoxicity (EspF and Cif), and host cell responses that ultimately generate watery diarrhea. Different from EPEC Tir, EHEC Tir is not tyrosine phosphorylated and does not interact with Nck in order to trigger actin polymerization at the site of bacterial adhesion; instead, EHEC translocates an effector protein, TccP/EspFu, which has an Nck-like activity and is essential to promote the reorganization of the actin cytoskeleton underneath adherent EHEC bacteria. ER, endoplasmic reticulum.

 

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  		TABLE 1. List of effector proteins of EHEC, EPEC, and C. rodentium

 
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 {alpha}-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 (Y474P); 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, Y474P-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).


   PROPHAGE-CARRIED EFFECTORS
 
In addition to the above-described LEE-encoded effectors, it is now evident that a number of effector proteins carried on prophages or other PAIs are translocated into the host cell via the LEE-encoded FTTSS. However, so far, only four of the newly identified effectors have been characterized; we summarize below our current understanding of their functions (Fig. 1 and 3; Table 1).

Cif. Cif (cycle inhibiting factor) was the first prophage-carried effector protein to be identified. It is carried on a {lambda} 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).


   ANOTHER EFFECTOR: EspG2
 
The third open reading frame (ORF3) within the espC PAI (Table 1) encodes a protein which shares 43.5% identity with LEE-encoded EspG and 19% identity with Shigella VirA (53). Present in EPEC (129), the ORF3 protein has been shown to be translocated (124, 153a) and to play a role similar to that of EspG; we therefore renamed the protein EspG2 (153a). EspG2 presents a clear example of effector functional redundancy (124).


   INTEGRATION OF EFFECTOR PROTEIN FUNCTIONS
 
(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/ß-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-{gamma}1, 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 {alpha}-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).


   CONCLUDING REMARKS
 
This review has been written at a time when the field of EPEC and EHEC studies is undergoing a minor revolution. Until April 2004, only five effectors were known. In December 2004, when we wrote this review, the number of known effectors (proven or deduced from the different genome projects) had risen to over 20. The discovery of TccP/EspFu has finally resolved the mystery of how EHEC induces actin polymerization in a tyrosine phosphorylation-/Nck-independent manner. At the same time, the use of human IVOC revealed the existence of a novel Tir Y474P-independent mechanism of EPEC-induced actin polymerization (Chong et al., unpublished results). Moreover, using human IVOC highlighted, for the first time, a role for an effector protein, EspF, in remodeling of the brush border microvilli, a poorly characterized aspect of EPEC and EHEC infection (152). However, despite the recent advances, there is still a long way to go until we fully discover and understand how EPEC and EHEC use their repertoire of effectors to inflict damage on host epithelial cells and to cause diarrhea.

Editor: J. B. Kaper


   ACKNOWLEDGMENTS
 
This work was supported by the Wellcome Trust.


   FOOTNOTES
 
* Corresponding author. Mailing address: CMMI, Flowers Building, Imperial College London, London SW7 2AZ, United Kingdom. Phone: (44) 20 7595 3070. Fax: (44) 20 7595 3069. E-mail: g.frankel{at}imperial.ac.uk. Back

{dagger} These authors contributed equally to this work. Back


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