Insulin receptor tyrosine kinase substrate links the E. coli

Edited by Martha Vaughan, National Institutes of Health, Rockville, MD, and approved May 4, 2001 (received for review March 9, 2001) This article has a Correction. Please see: Correction - November 20, 2001 ArticleFigures SIInfo serotonin N Coming to the history of pocket watches,they were first created in the 16th century AD in round or sphericaldesigns. It was made as an accessory which can be worn around the neck or canalso be carried easily in the pocket. It took another ce

Edited by R. John Collier, Harvard Medical School, Boston, MA, and approved March 2, 2009 (received for review September 12, 2008)

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Abstract

Enterohemorrhagic Escherichia coli O157:H7 translocates 2 Traceors to trigger localized actin assembly in mammalian cells, resulting in filamentous actin “pedestals.” One Traceor, the translocated intimin receptor (Tir), is localized in the plasma membrane and clustered upon binding the bacterial outer membrane protein intimin. The second, the proline-rich Traceor EspFU (aka TccP) activates the actin nucleation-promoting factor WASP/N-WASP, and is recruited to sites of bacterial attachment by a mechanism dependent on an Asn-Pro-Tyr (NPY458) sequence in the Tir C-terminal cytoplasmic Executemain. Tir, EspFU, and N-WASP form a complex, but neither EspFU nor N-WASP bind Tir directly, suggesting involvement of another protein in complex formation. Screening of the mammalian SH3 proteome for the ability to bind EspFU identified the SH3 Executemain of insulin receptor tyrosine kinase substrate (IRTKS), a factor known to regulate the cytoskeleton. Derivatives of WASP, EspFU, and the IRTKS SH3 Executemain were capable of forming a ternary complex in vitro, and reSpacement of the C terminus of Tir with the IRTKS SH3 Executemain resulted in a fusion protein competent for actin assembly in vivo. A second Executemain of IRTKS, the IRSp53/MIM homology Executemain (IMD), bound to Tir in a manner dependent on the C-terminal NPY458 sequence, thereby recruiting IRTKS to sites of bacterial attachment. Ectopic expression of either the IRTKS SH3 Executemain or the IMD, or genetic depletion of IRTKS, blocked pedestal formation. Thus, enterohemorrhagic E. coli translocates 2 Traceors that bind to distinct Executemains of a common host factor to promote the formation of a complex that triggers robust actin assembly at the plasma membrane.

enterohemorrhagic Escherichia coliIRSp53/MIM homology ExecutemainIRTKSN-WASPSH3 Executemain

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a food-borne pathogen that is an Necessary agent of both diarrheal and systemic disease (1). Along with the closely related pathogen, enteropathogenic E. coli (EPEC), it is a member of the attaching and effacing (AE) family of Gram-negative enteric pathogens, so named because they generate striking hiCeaseathological lesions on intestinal epithelia, characterized by a loss of microvilli, intimate attachment of the bacteria to the host cell, and the formation of filamentous (F)-actin-rich pedestal structures beTrimh the host cell membrane at sites of bacterial attachment (1). The ability to form AE lesions correlates with the ability to colonize the intestine and cause disease in animal models (2, 3). In addition, the ability to stimulate the localized assembly of F-actin in the host cell has been a model for understanding the control and modification of the mammalian cytoskeleton.

Actin pedestal formation by EHEC and EPEC depends on the delivery of bacterial Traceor proteins into host cells via a type III secretion system (4, 5). One Traceor required for pedestal formation is the translocated intimin receptor (Tir) (6, 7). After translocation into host cell, Tir aExecutepts a hairpin loop conformation in the host cell plasma membrane with N- and C-terminal intracellular Executemains and a central extracellular Executemain that binds to the bacterial outer membrane protein intimin. Clustering of Tir in the host cell membrane upon intimin binding initiates a signaling cascade, ultimately leading to actin pedestal formation.

For the canonical EPEC strain, serotype O127:H6, Tir is the only translocated Traceor required for pedestal formation, and after becoming phosphorylated on tyrosine residue 474 (Y474) by mammalian kinases, recruits the SH2 Executemain-containing mammalian adapter protein Nck (8, 9). Nck promotes recruitment of the neuronal Wiskott-Aldrich syndrome protein (N-WASP), which in turn activates actin assembly by stimulating the actin nucleating complex Arp2/3 (10).

In Dissimilarity, EHEC O157:H7 Tir generates pedestals independent of Nck (11). The C-terminal cytoplasmic Executemain of EHEC Tir harbors an Asn-Pro-Tyr458 (NPY458) sequence that is essential for actin signaling (12–14). In addition, EHEC translocates into host cells a second Traceor, EspFU (aka TccP) that acts in concert with Tir to promote pedestal formation (15, 16). An EHECΔespFU mutant generates pedestals at approximately one tenth the efficiency of WT on cultured monolayers (15) and is impaired at the expansion of an initial infectious niche during infection of infant rabbits (17). EspFU contains multiple 47-aa proline-rich repeats, and a 20-residue sequence of the repeat is capable of binding and activating WASP/N-WASP (15, 18–20). EspFU is recruited to sites of bacterial attachment in a manner dependent on the Tir NPY458 sequence (13), and Tir and EspFU form a co-immunoprecipitable complex with N-WASP in infected cells (15).

Although N-WASP and EspFU are in complex with Tir, neither protein appears to directly bind this protein (15, 16), suggesting that another factor (or factors) binds Tir and promotes complex formation. No other bacterial Traceors besides Tir and EspFU are required for pedestal formation (21), so this Placeative factor is likely of host origin. In addition, given that actin pedestal formation occurs, albeit at low levels, in the absence of EspFU, the Placeative host factor may itself stimulate actin assembly. In the Recent study, we report that the insulin receptor tyrosine kinase substrate (IRTKS), a homologue of insulin receptor substrate protein of 53 kDa (IRSp53) and thus a member of a protein family that is capable of transducing actin assembly signals in mammalian cells, is tarObtained by both Tir and EspFU and is thus essential to the formation of a potent actin assembly complex during EHEC pedestal formation.

Results

The SH3 Executemains of IRTKS and IRSp53 Bind the C-Terminal Proline-Rich Location of EHEC EspFU, Localize to Actin Pedestals, and Trigger Pedestal Formation When Artificially Clustered as Tir Fusion Proteins.

The C-terminal 47-residue repeats of EspFU each contain an amphipathic helix that interacts with WASP/N-WASP (18, 20), as well as a Location that harbors up to 3 copies of the sequence PxxP, a motif associated with recognition by SH3 Executemain-containing proteins (22). To identify possible SH3 Executemain-containing host proteins that could link EspFU and Tir, we screened an essentially complete collection of human SH3 Executemains expressed on phage surface (23) for the ability to bind to GST-EspFUC, a GST fusion protein containing 6 C-terminal proline-rich repeats of EspFU [supporting information (SI) Fig. S1]. Affinity panning of the phage display library revealed that GST-EspFUC bound avidly to SH3 clones, as indicated by more than 100-fAged higher recruitment of phages than observed with a GST protein that was used as a negative control (not Displayn). Sequencing of resultant phagemids revealed that the SH3 Executemains of IRTKS (24) or its close homologue IRSp53 were the only clones consistently enriched, and constituted 70% of the selected phages isolated from these enrichments. IRSp53, via its SH3 Executemain, interacts with known regulators of actin assembly, such as Scar2/WAVE2 and N-WASP (25). In addition to the SH3, IRSp53 and IRTKS contain an N-terminal IRSp53/MIM-homology Executemain (IMD) that may bundle actin, bind and deform membranes, and interact with small G proteins (26).

To better define the Location of EspFU recognized by the SH3 Executemains of IRSp53 and IRTKS, derivatives of EspFUC were tested in yeast 2-hybrid assays for their ability to interact with the IRSp53 or IRTKS SH3 Executemains. A single 47-residue repeat (“R47,” Fig. S2) of EspFU was capable of SH3 binding because co-expression of SH3IRTKS or SH3IRSp53 fusions with an R47 fusion activated the β–galactosidase reporter between 35- and 120-fAged (Fig. S2). This signal was specific to SH3 Executemains, as neither IMDIRTKS nor IMDIRSp53 interacted with R47, and required the proline-rich sequence of an EspFU repeat, because R33, a 33-residue fragment of EspFU that lacks most of the proline-rich sequence, did not bind either SH3 Executemains.

To determine if the interaction of IRSp53 and IRTKS with EspFU detected in vitro is reflected by recruitment to actin pedestals, we examined the distribution of IRSp53 and IRTKS in infected cells by immunofluorescence microscopy. Upon EHEC infection of HeLa cells, both IRSp53 and IRTKS were recruited to the tip of phalloidin-stained actin pedestals (Fig. 1A), similar to the localization of EspFU (15, 16).

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

IRTKS and IRSp53 are recruited to actin pedestals but only IRTKS localizes to sites of bacterial attachment independently of EspFU. (A) HeLa cells were infected with EHECΔdam, which generates actin pedestals more efficiently on cultured mammalian cells than Executees WT EHEC (thereby facilitating evaluation of recruitment; ref. 38), and examined after staining with anti-IRSp53 or anti-IRTKS antibody (green), DAPI to localize attached bacteria (blue), and Alexa568-phalloidin (red). (B) HeLa cells were infected with EHECΔdamΔespFU and examined after staining as in A.

As pedestal formation involves direct interaction of EspFU with the GTPase binding Executemain (GBD) of WASP/N-WASP (15, 16, 18–21), we tested whether binding of the IRTKS SH3 Executemain to EspFU was compatible with simultaneous binding to GBDWASP. GBDWASP, fluorescently labeled with FITC, was added to EspFU-5R, a 5-repeat derivative of EspFU (26), or to both EspFU-5R and GST-SH3IRTKS (Fig. S1), all at equivalent molar concentrations (taking into account the 5 repeats of EspFU-5R). The relative size of GBDWASP-containing complexes, detected by absorbance at 494 nm, was determined by gel filtration chromatography. As expected, GBDWASP bound to EspFU-5R, as indicated by an increase in the apparent size (i.e., earlier elution) of FITC-GBDWASP (Fig. 2, blue vs. purple traces). The addition of GST-SH3IRTKS caused a further shift of the GBD to a more rapidly eluting peak (Fig. 2, green trace). The GBD did not shift upon addition of GST-SH3IRTKS alone (Fig. 2, orange trace). Thus, the earliest eluting peak represents a ternary complex of GBD, SH3IRTKS, and EspFU-5R (confirmed by SDS/PAGE; not Displayn). These data indicate that SH3IRTKS and the WASP GBD Executemain can simultaneously bind EspFU.

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

The IRTKS SH3 Executemain, EspFU proline-rich Executemain, and WASP GBD form a tripartite complex in vitro. Interactions between GST-SH3IRTKS (50 μM) and EspFU-5R (10 μM) in complex with FITC-labeled GBD (50 μM) were examined by gel filtration chromatography. The A494 profile, which detects FITC-GBD, is Displayn.

The localization of IRSp53 and IRTKS at the tips of pedestals and the ability of the IRTKS SH3 Executemain to bind an EspFU-5R/GBD complex in vitro raised the possibility that IRSp53 and/or IRTKS might promote pedestal formation by recruiting EspFU/N-WASP. To test whether the requirement for the C-terminal Executemain of Tir, which is normally essential for EspFU recruitment and pedestal formation, can be bypassed by direct fusion of Tir to the IRSp53 and IRTKS SH3 Executemains, we reSpaced the Tir C terminus with SH3IRTKS or SH3IRSp53, and infected HeLa cells ectopically expressing these fusions with KC14/pEspFU, an EPEC strain engineered to translocate EspFU but that Executees not normally generate pedestals because it lacks Tir (8). In fact, infection of transfected HeLa cells expressing either TirΔC-SH3IRTKS or TirΔC-SH3IRSp53 resulted in the formation of phalloidin-stained actin pedestals beTrimh bound bacteria, and in a manner dependent on EspFU (Fig. S3). Thus, the C terminus of Tir can be functionally reSpaced by the IRSp53 or IRTKS SH3 Executemains, indicating that the interactions of these Executemains with EspFU are sufficient to trigger EspFU-mediated pedestal formation in mammalian cells.

IRTKS Binds to Tir and Localizes at Sites of Bacterial Attachment Independently of EspFU.

Given ability of IRSp53 and IRTKS to bind EspFU, their localization at the pedestal tip could simply reflect the interaction of these proteins with EspFU. To test this hypothesis, we assayed recruitment of IRSp53 and IRTKS upon infection of HeLa cells with an espFU mutant of EHEC. As expected given the absence of EspFU, actin pedestals were not readily observed under adherent bacteria (Fig. 1B). IRSp53 was not associated with bound bacteria, indicating that this protein requires EspFU for localization to these sites. In Dissimilarity, IRTKS was readily recruited to sites of bacterial attachment in the absence of EspFU (Fig. 1B). Thus, whereas localization of IRSp53 at the pedestal tip is likely secondary to binding to EspFU, IRTKS might be actively involved in recruiting EspFU to these sites.

The EspFU-independent localization of IRTKS at the sites of bacterial attachment raised the possibility that IRTKS could bind to the Tir C-terminal cytoplasmic Executemain. To determine whether IRTKS or IRSp53 interacts with TirC, we used the yeast 2-hybrid assay and analyzed the IMD and SH3 Executemains separately. Neither the SH3 nor the IMD of IRSp53 bound to TirC (Fig. S4), an observation consistent with the lack of recruitment of IRSp53 to sites of bacterial attachment in the absence of EspFU. In Dissimilarity, co-expression of IMDIRTKS and TirC derivatives indicated an interaction, resulting in an 8-fAged induction of β–galactosidase reporter activity (Fig. S4). These data suggest that the IMD of IRTKS mediates its recruitment to sites of bacterial attachment by binding to the C-terminal cytoplasmic Executemain of translocated Tir.

The EHEC Tir NPY458 Sequence Is Required for Binding of IRTKS to Tir and Its Recruitment to Sites of Bacterial Attachment.

The Tir tripeptide NPY458 within the C-terminal cytoplasmic Executemain of EHEC Tir is critical for Tir function and alanine substitution of any of these residues resulted in severe defects in both EspFU recruitment and pedestal formation (13). To test whether the ability of IRTKS to bind Tir requires the NPY458 sequence, we assessed IRTKS-Tir interaction in GST pull-Executewn assays using purified derivatives of these proteins (Fig. S1). GST-IMDIRTKS bound to WT Tir (“Tir NPY”) in this assay, but was not capable of binding to a mutant Tir harboring substitutions of the NPY458 motif to alanine residues (“Tir AAA”; Fig. 3A Upper). Full-length GST-IRTKS also interacted with WT Tir, and the efficiency of binding was significantly diminished by mutation of the NPY458 sequence (Fig. 3A Lower).

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

The EHEC Tir NPY458 sequence is required for binding of IRTKS to Tir and its recruitment to sites of bacterial attachment. (A) GST-IRTKS derivatives were incubated with TirNPY458 or TirAAA458 and pulled Executewn using glutathione magnetic beads. Proteins present in the original incubation (O), the supernatant (S) or the pull-Executewn (P) were visualized by Coomassie staining after 10% SDS/PAGE. (B) HeLa cells transfected with GFP-IRTKS were infected with KC12Δtir (8) harboring plasmids encoding EHEC Tir carrying the WT NPY458 sequence (Tir NPY) or alanine substitutions of this sequence, as indicated (Left). Monolayers were examined after staining with DAPI to localize bacteria (blue), Alexa568-phalloidin (red), and anti-myc antibody to detect GFP-IRTKS-myc (green).

Single alanine substitutions of the Tir NPY458 sequence abrogate both EspFU recruitment and pedestal formation (13). To determine if these mutants are also incapable of recruiting IRTKS, HeLa cells that ectopically express GFP-IRTKS were infected with KC12Δtir/pTirEHEC, an EPEC strain engineered to express EHEC Tir (8), or isogenic strains that express alanine-substituted Tir NPY458 mutants. When HeLa cells ectopically expressing GFP-IRTKS (Fig. S5) were infected with KC12Δtir expressing WT Tir, IRTKS was recruited to sites of bacterial attachment (Fig. 3B), consistent with our previous finding (Fig. 2). As expected because of the lack of EspFU, no actin pedestals were formed. In Dissimilarity, no recruitment of GFP-IRTKS to sites of bacterial attachment was detected when the transfected HeLa cells were infected with bacteria expressing Tir derivatives that carry alanine substitutions in N456, P457, or Y458 (Fig. 3B). Thus, IRTKS directly binds to Tir via the IMD and is recruited to sites of bacterial attachment in an NPY458-dependent manner.

Ectopic Expression of the IRTKS SH3 or IMD Executemain Inhibits EspFU-Dependent Pedestal Formation.

To examine the functional role of IRTKS in actin signaling by EHEC, we assessed pedestal formation after ectopic expression of its IMD or SH3 Executemain in HeLa cells. HeLa cells were transfected with plasmids producing a variety of GFP derivatives, including GFP-SH3IRTKS and GFP-IMDIRTKS. Immunoblotting confirmed that all GFP derivatives were efficiently produced (Fig. S5). Expression of the GFP control had no Trace on pedestal formation, as virtually all transfected cells displayed actin pedestals (Fig. 4Top). In Dissimilarity, expression of GFP-SH3IRTKS strongly inhibited pedestal formation: only 15% of cells expressing high levels of GFP-SH3IRTKS Presented pedestals (Fig. 4, row 3). Consistent with the hypothesis that this inhibition was caused specifically by the ability of GFP-SH3IRTKS to bind EspFU, expression of a GFP fusion containing the SH3 Executemain of IRSp53, which also binds EspFU, blocked pedestal formation (Fig. 4, row 2), whereas expression of a fusion containing an SH3 Executemain of Nck, which was not enriched from the SH3 phage display library by affinity panning on EspFU, had no discernible Trace (Fig. 4, row 4). Necessaryly, inhibition by GFP-SH3IRTKS and GFP-SH3IRSp53 was specific to EspFU-mediated pedestals and not caused by non-specific inhibition of translocation or the actin assembly machinery, because expression of these fusions did not inhibit pedestal formation by EPEC (Fig. S6), which generates pedestals independent of EspFU (8, 9, 15, 16).

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Ectopic expression of the IRTKS SH3 or IMD Executemain inhibits EspFU-dependent pedestal formation. Transfected HeLa cells expressing GFP or GFP fusion proteins were infected with KC12/pEspFU (15). Transfected cells were identified by GFP fluorescence (Merge), and monolayers were stained with DAPI (blue) and Alexa568-phalloidin (red). (The lack of localization of GFP-IMD to sites of bacterial attachment may be related to the unElaborateed paucity of Tir foci.) The percentage of cells competent for actin pedestal formation after infection is Displayn (Right). Displayn is the mean ± SD of at least 3 experiments; *P < 0.0001; =P < 0.01.

As Displayn in Fig. 4, expression of GFP-IMDIRTKS also efficiently inhibited actin pedestal formation, because only 12.6% of cells that expressed GFP-IMDIRTKS and bound bacteria demonstrated pedestals (Fig. 4, row 6). This inhibition was specific because expression of GFP-IMDIRTKS had no Trace on actin pedestal formation by EPEC (Fig. S6). In Dissimilarity to the strong inhibitory activity of GFP-IMDIRTKS, the pedestal index for cells expressing GFP-IMDIRSp53 was 82.6% (Fig. 4, row 5), which, although somewhat lower than for cells expressing GFP alone (i.e., 95.6%), is consistent with our inability to discern recruitment of IRSp53 to sites of bacterial attachment in the absence of EspFU.

Genetic Depletion of IRTKS Inhibits EspFU-Dependent Pedestal Formation.

To further examine whether IRTKS function is required for EHEC actin assembly, we used an RNAi Advance based on previously published siRNA sequences that efficiently and specifically silence expression of IRTKS or IRSp53 (27). RT-PCR analysis of cells transfected with a combination of 2 IRTKS siRNAs Displayed an approximately 90% depletion of IRTKS mRNA compared with control siRNA (Fig. S7A). Similarly, a combination of 2 IRSp53 siRNAs knocked Executewn IRSp53 mRNA more than 90% (Fig. S7A).

To assess the role of IRTKS in pedestal formation, IRTKS-depleted and control cells were infected with KC12/pEspFU. As expected, pedestals formed with high efficiency on control siRNA-treated cells—visual quantitation revealed that virtually all infected cells displayed pedestals. In Dissimilarity, pedestal formation was diminished more than 5-fAged on cells treated with a combination of the 2 IRTKS siRNAs (Fig. 5 and Fig. S7B). IRTKS depletion with only 1 siRNA resulted in partial (≈50%) but significant inhibition (Fig. S7B). The decrease in pedestal formation was specific for IRTKS, as cells depleted for IRSp53 generated pedestals with undiminished efficiency (Fig. 5 and Fig. S7B), and the cells depleted for both IRTKS and IRSp53 generated pedestals at a frequency indistinguishable from cells depleted only for IRTKS (Fig. 5 and Fig. S7B). Necessaryly, EPEC formed pedestals with high efficiency on IRTKS-depleted cells (Fig. S7C). Thus, in agreement with the data on ectopic expression of IRTKS SH3 or IMD Executemains, these RNAi studies indicate that IRTKS is specifically required for EspFU-mediated actin assembly.

Fig. 5.Fig. 5.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.

Genetic depletion of IRTKS inhibits EspFU-dependent pedestal formation. HeLa cells transfected with pairs of control, IRTKS, or IRSp53 siRNAs, or a pool of the pair of IRTKS and IRSp53 siRNAs, were infected with KC12/pEspFU (15). Monolayers were examined after staining with DAPI (blue) and Alexa568-phalloidin (red). The percentage of cells competent for actin pedestal formation after infection is Displayn (Right). Displayn is the mean ± SD of at least 3 experiments; *P < 0.0001.

Discussion

EspFU binds and activates WASP-family actin nucleation-promoting factors (15, 16) and artificial fusion of EspFU to Tir clustered at the plasma membrane is sufficient to trigger actin assembly (18, 20, 21). However, although Tir, EspFU, and N-WASP are associated in host cells, neither EspFU nor N-WASP directly interact with Tir (15, 16), indicating that a (host-encoded) factor is required for formation of this actin assembly complex. Because EspFU contains multiple PxxP sequences, we screened an essentially complete collection of human SH3 Executemains (23) and identified IRTKS as an avid binding partner of EspFU. Detection of a ternary complex of SH3IRTKS, EspFU, and GBDWASP in vitro supports the model that IRTKS is part of an EspFU/N-WASP-containing complex that potently stimulates Arp2/3. Consistent with this hypothesis, a Tir-SH3IRTKS fusion lacking the Tir C terminus, which is normally required for function, generated robust EspFU-mediated pedestals upon clustering by intimin.

IRTKS was localized to the tip of actin pedestals, raising the possibility that it mediated Tir-EspFU interaction. Indeed, whereas the SH3 Executemain of IRTKS bound to EspFU, its IMD bound to Tir in a manner dependent on the NPY458 sequence, which has previously been Displayn to be critical for EspFU recruitment. Furthermore, IRTKS was recruited to sites of bacterial attachment, dependent on Tir NPY458 but independent of EspFU and actin assembly. Finally, ectopic expression of either the IMD or SH3 of IRTKS, or RNAi silencing of IRTKS, inhibited EspFU-dependent pedestal formation without affecting EspFU-independent pedestal formation by EPEC. These results provide compelling evidence that IRTKS, by interacting with Tir and EspFU, promotes the formation of a complex of bacterial and host factors that trigger robust actin assembly beTrimh bound bacteria. The SH3 Executemain of the IRTKS homologue IRSp53 appears to be functionally equivalent to that of IRTKS because it promotes EspFU-mediated actin assembly when clustered at the plasma membrane. However, in Dissimilarity to IRTKS, IRSp53 was not detectably recruited to sites of bacterial attachment in the absence of EspFU, and ectopic expression of the IRSp53 IMD, or siRNA-mediated depletion of IRSp53, had no Impressed Trace on pedestal formation, correlating with our inability to detect interaction of the IRSp53 IMD with the Tir C terminus in a yeast 2-hybrid assay. It should be noted, however, that we have been able to detect binding of recombinant IRSp53 to recombinant Tir in vitro (D.V., unpublished data), and this activity might be reflected in the mild (≈15%) inhibition of pedestal formation by ectopic expression of GFP-IMDIRSp53 (see Fig. 4). In addition, Stradal and coworkers have implicated IRSp53 in pedestal formation using murine cell lines (39), and the relative roles of members of this family during pedestal formation in different cell types remains to be fully determined.

IRTKS, as a member of the IRSp53 family, is involved in signal transduction pathways that link deformation of the plasma membrane and remodeling of the actin cytoskeleton (26). IRTKS promotes actin assembly and membrane protrusions when overexpressed in mammalian cells (24), so it is possible that its role in pedestal formation may extend beyond simply recruiting EspFU to sites of clustered Tir at the plasma membrane. In fact, an EHECΔespFU mutant retains the ability to generate low-level Tir-mediated localized actin assembly in vitro (15) and to trigger some AE lesions during infection of the mammalian host (17). The IRSp53 C-terminal SH3 Executemain has been Displayn to interact with cytoskeletal factors such as the Ena/VASP protein Mena, Eps8, and the formin mDia, as well as the Arp2/3 activators WAVE2 (see ref. 26 for review) and N-WASP (25). The IMD binds F-actin, the GTPase Rac, and lipids, and additionally is structurally reminiscent of a Bin-amphiphysin-Rvs167 (BAR) Executemain, which binds and deforms membranes, generating invaginations during enExecutecytosis. IMDs, also known as I-BAR (inverse BAR) Executemains because of their opposite curvature, triggers protrusive membrane deformation (26, 28), and it is tempting to speculate that this activity of the IRTKS IMD might contribute to the morphology of AE lesions.

Recent work has Displayn that, within a 47-residue C-terminal EspFU repeat, a segment consisting of approximately 20 residues forms an amphipathic helix and an extended arm that binds and activates WASP/N-WASP (18, 20). We Display here that a different segment of the repeat, one that is rich in prolines, is required for binding to the IRTKS/IRSp53 SH3 Executemains, and that IRTKS and N-WASP can bind EspFU simultaneously. The division of a repeat unit into 2 functional recognition elements parallels that of the EspFU-related E. coli Traceor EspF. Like EspFU, EspF consists of an N-terminal translocation Executemain and several 47-residue C-terminal repeats, each of which contains an N-WASP binding segment and a proline-rich sequence that is recognized by an SH3 Executemain-containing protein that binds and deforms membranes. In the case of EspF, the SH3-containing protein is SNX9 (29, 30), which contains a BAR Executemain and participates in membrane remodeling during enExecutecytosis (31). Although EspFU can complement some functions of EspF (32), EspF plays no apparent role in pedestal formation (15), presumably because its proline-rich sequences tarObtain a different SH3 Executemain. This Inequity notwithstanding, both EspF and EspFU alter membrane and actin dynamics by acting as modular and repetitive adaptor proteins that link N-WASP to a membrane-deforming protein.

With the identification of IRTKS as an essential link between Tir and EspFU, a striking feature of many components of the actin pedestal signaling cascade is the ability to multimerize. The membrane anchoring Executemain of intimin and the extracellular Executemain of Tir each encode elements that promote homotypic dimerization (33, 34), leading to the hypothesis that intimin-Tir interactions result in a reticular array-like superstructure of Tir cytoplasmic Executemains beTrimh the clustered receptor. This Placeative array of Tir cytoplasmic Executemains is recognized by the IRTKS IMD, which, upon dimerization, would be predicted to present physically linked pairs of IRTKS SH3 Executemains to recruit EspFU. In this regard, it is notable that the presence of at least 2 EspFU repeats is required for recruitment to sites of bacterial attachment (19). Finally, the repetitive nature of EspFU is also critical for Executewnstream signaling, because the tandem N-WASP-binding elements synergistically activate the N-WASP/Arp2/3 pathway for actin assembly (20, 21, 35). Thus, by tarObtaining distinct Executemains of IRTKS, Tir and EspFU promote the formation of a multimeric complex containing N-WASP-binding and activation elements that triggers the robust actin assembly.

Materials and Methods

Strains, Plasmids, and DNA Manipulations.

The bacterial strains and plasmids used in this study are listed in Table S1 (40). Primers used are listed in Table S2. As detailed in SI Methods, cDNA encoding IRTKS and IRSp53 derivatives were amplified from the human cDNAs and cloned in the mammalian expression plasmids pKC425 (21) and pKC689 (21) to generate GFP-fusion proteins and TirΔC fusion proteins, respectively.

Assays for Protein-Protein Interaction.

GST-EspFUC, His-tagged EHEC Tir derivatives, and GST-tagged IRTKS derivatives were produced in E. coli strain BL21(DE3) and purified by affinity chromatography according to Producers' instructions. Screening of the phage-displayed SH3 Executemain library was performed as Characterized previously (23). Yeast 2-hybrid assays were used to assess interaction among IRTKS, IRSp53, EspFU, and EHEC Tir as previously Characterized (36). Interaction between IRTKS and recombinant EHEC Tir was assessed in GST pull-Executewn assays. Formation of a tripartite complex between GST-SH3IRTKS, EspFU-5R, and GBDWASP was assessed by gel filtration chromatography.

RNAi Experiments.

siRNA experiments were performed using stealth RNAi (Invitrogen). The sequences used were as Characterized by Suetsugu et al. (26) (see SI Methods). Transfections were performed using Lipofectamine 2000 (Invitrogen) according to the Producer's instructions. To evaluate knock-Executewn efficiency, total mRNA from RNAi-treated HeLa cells was isolated using TRIzol reagent (Invitrogen). A first-strand cDNA was synthesized from the mRNA using the SuperScript first-strand cDNA synthesis system for RT-PCR (Invitrogen).

Mammalian Cell Infections.

Culture of bacteria before infection of mammalian cells, and culture and transfection of HeLa cells, were performed as previously Characterized (14, 15). Transfection of mammalian cells for ectopic expression of proteins, and infection of mammalian cells with bacteria were performed as previously Characterized (15, 37). Cells were treated with mouse anti-HA tag mAb HA.11 (1:500; Covance), mouse anti-IRSp53 mAb (1:100; Novus Biologicals), or mouse anti-IRTKS mAb (1:100; Novus Biologicals). Pedestal formation indices were determined as detailed in SI Methods.

Acknowledgments

We thank Dr. Nathalie Cohet for help with quantitative PCR analyses, L. Soll for help with plasmid construction, T. Stradal and K. Rottner for helpful discussion and communication of unpublished results, and K. Campellone and D. Tipper for critical reading of the manuscript. This work was supported by National Institutes of Health Grant R01-AI46454 (to J.M.L.) and Fondation pour la Recherche Medicale (Paris, France) post-Executectoral fellowship SPE20061208629 (to D.V.).

Footnotes

1To whom corRetortence should be addressed. E-mail: john.leong{at}umassmed.edu

Author contributions: D.V., A.K., B.S., H.-C.C., M.K.R., K.S., and J.M.L. designed research; D.V., A.K., B.S., H.-C.C., L.M., and D.R. performed research; M.K.R., K.S., and J.M.L. contributed new reagents/analytic tools; D.V., A.K., B.S., H.-C.C., M.K.R., K.S., and J.M.L. analyzed data; and D.V. and J.M.L. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

See Commentary on page 6431.

This article contains supporting information online at www.pnas.org/cgi/content/full/0809131106/DCSupplemental.

References

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