Tails of two Tirs: actin pedestal formation by enteropathogenic E. coli and enterohemorrhagic E. coli O157:H7
Introduction
Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli O157:H7 (EHEC) are diarrheagenic human pathogens belonging to a distinct family of enteric bacteria that form unique structures called attaching and effacing (AE) lesions on the surfaces of intestinal epithelial cells. AE lesions are characterized by the loss of microvilli, an intimate adherence of bacteria adjacent to the host cell membrane, and the generation of organized cytoskeletal structures containing filamentous actin beneath sites of bacterial attachment, termed actin pedestals (Figure 1a). The formation of actin pedestals by EPEC and EHEC is recapitulated on cultured mammalian cells (Figure 1b–d), and the ability of AE organisms to form pedestals on cultured cells correlates with their ability to colonize the intestine and cause disease in human and other animal hosts.
EPEC and EHEC each contain a highly homologous 35 kb chromosomal pathogenicity island called the locus of enterocyte effacement (LEE) 1., 2., which contains genes critical for pedestal formation. The LEE encodes a type III protein secretion system for the contact-dependent translocation of bacterial proteins into host cells. Effector proteins that are transported through the type III secretion apparatus are also encoded within the LEE. Translocated proteins that have been identified to date are the E. coli secreted proteins EspA, EspB, EspD, EspF and EspG, Map (mitochondria-associated protein), and the translocated intimin receptor (Tir/EspE) (Figure 2a). EspA forms a filamentous structure on the bacterial surface to contact host cells, through which other LEE-encoded effectors are secreted, including EspB and EspD 3., 4., 5.. EspB and EspD are thought to transit through the EspA filament to form a pore, or translocon, in the host plasma membrane, to deliver other virulence factors into the cell 6., 7., 8., 9., 10. and perhaps act as translocated effectors themselves 11., 12., 13.. EspF, EspG and Map affect other host cell processes but do not play an observable role in actin pedestal formation 14., 15., 16., 17.. A functional EspA conduit and EspB/D translocon are required for translocation of Tir, the best-characterized bacterial effector and the one that plays the central role in triggering actin pedestal formation by the host cell.
Our conception of how actin pedestals are formed was transformed by the discovery that EPEC and EHEC inject Tir into the host plasma membrane, where it functions as a bacterial receptor 18., 19.. In the plasma membrane, Tir adopts a hairpin-loop structure featuring a central extracellular domain that binds to the LEE-encoded outer membrane protein intimin 20., 21., 22. (Figure 2b). Intimin may also promote initial adherence by binding to endogenous host cell receptors 23., 24., but this aspect of intimin will not be discussed here. The amino- and carboxy-terminal domains of Tir reside in the host cell cytoplasm 25., 26., 27., where they are capable of interacting with host cytoskeletal and signaling components (Figure 2b). After translocation of Tir and other Esps, the interaction between Tir and intimin is sufficient to initiate actin assembly, because intimin-coated beads form pedestals on mammalian cells pre-infected with an EPEC strain that does not express intimin but does translocate Tir and other effectors [28]. The key role of this interaction is emphasized by the observation that intimin point mutations that reduce Tir binding activity in vitro exhibit similarly diminished abilities to trigger pedestal formation [22]. Thus, in addition to serving as a bacterial adhesion receptor, Tir accomplishes the added function of exploiting actin signaling cascades within host cells upon its interaction with intimin. In this review we discuss the mechanisms by which EPEC and EHEC Tir exploit the mammalian actin assembly machinery.
Section snippets
Microbial pathogens target the Arp2/3 pathway of actin assembly
A critical controller of actin polymerization at the eukaryotic plasma membrane is the heptameric actin-related protein 2/3 (Arp2/3 complex) that is capable of promoting actin nucleation 29., 30.. The actin nucleating activity of the Arp2/3 complex can be stimulated by its interaction with Wiskott–Aldrich syndrome protein (WASP) family members, such as neuronal (N-)WASP, the most widely expressed member of this family of proteins. The ability of N-WASP to stimulate the Arp2/3 complex can, in
Divergence in Tir-based actin signaling
EPEC and EHEC each use highly homologous gene products encoded on their LEE elements to generate pedestals that appear morphologically similar. However, in part owing to its ability to form pedestals at a higher efficiency in vitro [41], EPEC rather than EHEC has been preferentially used in studies on actin assembly, and in many ways has been regarded as a model for EHEC pedestal formation. Nevertheless, detailed analyses of the processes that EPEC and EHEC employ to assemble actin have
A protein that interacts with EPEC Tir but not EHEC Tir: the Nck adaptor
A breakthrough in deciphering the function of this segment of EPEC Tir occurred when two laboratories recognized that it was highly homologous to Nck-binding sequences, particularly the region surrounding Tyr112 of the vaccinia virus A36R protein 49.••, 50.••. Phosphotyrosine 112 has previously been implicated in Nck recruitment and actin-tail formation by vaccinia [35], suggesting that EPEC Tir similarly recruits Nck during pedestal formation.
In fact, two recent studies demonstrate that Tir
Signaling downstream of Nck
The identification of Nck binding as a proximal and essential interaction in actin pedestal formation by EPEC was a seminal finding, leading to straightforward models for EPEC actin signaling (Figure 3a). Nck can directly stimulate N-WASP to activate the Arp2/3 complex in vitro [32], suggesting that after recruitment by tyrosine-phosphorylated Tir, Nck might simply bind and activate N-WASP to trigger actin assembly. Alternatively, Nck binding by EPEC Tir might indirectly recruit N-WASP during
Management of Nck signaling
Molecules that might modulate activation of N-WASP by Nck also deserve consideration, as these simple models are developed further. For example, phosphatidylinositol 4,5-bisphosphate (PI[4,5]P2) and Nck synergistically activate N-WASP in vitro [32]. PI(4,5)P2 can accumulate in lipid rafts, which serve as platforms for the activation of signaling components at the plasma membrane 52., 53.. Interestingly, several raft-associated proteins, such as CD44 and annexin-2, as well as
Potential roles of other actin-associated molecules in pedestal formation
The composition of the actin pedestal is extremely complex (Table 1), and many of its constituents besides Nck, N-WASP and Arp2/3 might play important roles. For example, cortactin, which has the ability to stimulate Arp2/3 in a manner similar to N-WASP [55], localizes to sites of bacterial adherence in a Tir-independent manner (Table 1). Moreover, overexpression of a fragment of cortactin incapable of stimulating the Arp2/3 complex impairs the generation of EPEC pedestals [56]. The actin
The enteropathogenic E. coli Tir–Nck interaction is sufficient to initiate actin assembly
Although the identification of Nck as a signaling molecule acting directly downstream of Tir clearly narrowed the universe of plausible models of actin-pedestal formation by EPEC, several aspects of this process have complicated the identification of the minimal elements critical for actin assembly. For example, it was not clear if other translocated EPEC molecules were essential for actin condensation. Seven EPEC-secreted proteins have been identified so far, and it has been difficult to
Nck-independent pedestal formation by enterohemorrhagic E. coli O157:H7
Compared with the relative simplicity of actin assembly by EPEC, pedestal formation by EHEC is more complex and less well characterized. EHEC generates pedestals independently of Nck, because infecting EHEC do not recruit Nck to sites of actin polymerization (Figure 1d; Table 1) and can form pedestals on cell lines that do not express Nck [50••]. DeVinney et al. [47••] demonstrated that Nck-independent actin signaling by EHEC requires translocation of one or more bacterial factors in addition
Conclusions and future work
Through the years, the vast majority of studies designed at elucidating the mechanism of actin-pedestal formation have focused on EPEC and its effectors. Until recently, the manner by which EHEC and EPEC formed pedestals was assumed to be virtually identical, because the LEE pathogenicity islands were highly conserved and the pedestals appeared morphologically similar. However, recent studies clearly dictate that these two pathogens have evolved different Tir-based schemes to usurp host-cell
References and recommended reading
Papers of particular interest, published within the annual period of review, have been highlighted as:
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of special interest
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of outstanding interest
Acknowledgements
We thank Jenifer Coburn, Jon Goguen, Nikhat Parveen, Susannah Rankin and Donald Tipper for helpful discussion and careful review of the manuscript, Scott Snapper for providing unpublished results, and Saul Tzipori and Stuart Knutton for providing micrographs. This work was supported by the National Institutes of Health (grant NIH-R01–AI46454 to JM Leong).
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