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Infection and Immunity, October 2008, p. 4669-4676, Vol. 76, No. 10
0019-9567/08/$08.00+0     doi:10.1128/IAI.00140-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Cortactin Recruitment by Enterohemorrhagic Escherichia coli O157:H7 during Infection In Vitro and Ex Vivo

Aurelie Mousnier,^1, Andrew D. Whale,^1, Stephanie Schüller,² John M. Leong,³ Alan D. Phillips,² and Gad Frankel^1*

Division of Cell and Molecular Biology, Imperial College London,¹ Centre for Paediatric Gastroenterology, Royal Free and University College Medical School, London, United Kingdom,² Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester, Massachusetts³

Received 31 January 2008/ Returned for modification 7 March 2008/ Accepted 21 July 2008

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Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an important human pathogen that colonizes the gut mucosa via attaching and effacing (A/E) lesions; A/E lesion formation in vivo and ex vivo is dependent on the type III secretion system (T3SS) effector Tir. Infection of cultured cells by EHEC leads to induction of localized actin polymerization, which is dependent on Tir and a second T3SS effector protein, TccP, also known as EspF_U. Recently, cortactin was shown to bind both the N terminus of Tir and TccP via its SH3 domain and to play a role in EHEC-triggered actin polymerization in vitro. In this study, we investigated the recruitment of cortactin to the site of EHEC adhesion during infection of in vitro-cultured cells and mucosal surfaces ex vivo (using human terminal ileal in vitro organ cultures [IVOC]). We have shown that cortactin is recruited to the site of EHEC adhesion in vitro downstream of TccP and N-WASP. Deletion of the entire N terminus of Tir or replacing the N-terminal polyproline region with alanines did not abrogate actin polymerization or cortactin recruitment. In contrast, recruitment of cortactin to the site of EHEC adhesion in IVOC is TccP independent. These results imply that cortactin is recruited to the site of EHEC adhesion in vitro and ex vivo by different mechanisms and suggest that cortactin might have a role during EHEC infection of mucosal surfaces.

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Enterohemorrhagic Escherichia coli (EHEC), particularly serotype O157:H7, is an important human pathogen that adheres closely to the gut epithelium and triggers localized effacement of the brush border microvilli, a phenomenon known as attaching and effacing (A/E) (19). A/E lesion formation follows translocation of effector proteins, notably Tir/EspE (9, 18), through a type III secretion system (T3SS) (reviewed in reference 12). Following translocation, Tir is integrated into the enterocyte plasma membrane in a hairpin topology (15); the surface-exposed loop functions as the receptor for the outer membrane bacterial adhesin intimin (reviewed in reference 11).

During infection of epithelial cells in culture, EHEC triggers localized actin polymerization at the site of bacterial attachment (reviewed in references 5, 10, and 16), an activity completely dependent on Tir. EHEC also encodes a second T3SS bacterial effector protein, TccP, also known as EspF_U, which binds and activates the neuronal Wiskott-Aldrich syndrome protein (N-WASP), leading to actin polymerization via the actin-related protein 2/3 (Arp2/3) complex (6, 13). Tir and TccP are necessary and sufficient for robust actin polymerization in vitro because transfection of Tir and TccP and artificial clustering of Tir by nonpathogenic E. coli K-12 expressing intimin or a T3SS EHEC mutant triggers actin polymerization (K. Campellone, unpublished data). To date, no direct interaction between Tir and TccP has been reported. It is therefore plausible that an adaptor protein encoded by the mammalian cell links the two bacterial proteins. Recruitment of TccP to Tir_EHEC is mediated by a C-terminal tripeptide NPY₄₅₈ motif (3), suggesting that this sequence could be a target of a putative host adaptor.

Although robust pedestal formation in vitro requires TccP, an EHEC O157:H7 tccP mutant can trigger inefficient actin polymerization on cultured monolayers (4), suggesting that EHEC triggers multiple pathways of actin assembly in host cells. Consistent with this, EHEC O157:H7 tccP mutants can induce A/E lesions on ex vivo human intestinal organ cultures (13) and in animal models (22, 26). These lesions do not appear dramatically different from A/E lesions formed by wild-type EHEC, and it is possible that alternative pathways leading to A/E lesion formation function at a higher level during infection of primary intestinal epithelium than during infection of immortalized cell lines.

The actin pedestals of EHEC O157:H7 are complex structures that are enriched with focal adhesion (e.g., -actinin and talin), cytoskeletal (e.g., actin and cytokeratins 8 and 18), endocytic (e.g., dynamin 2), and actin assembly-regulating (e.g., N-WASP, Arp2/3, and cortactin) proteins (10, 14, 16). Cortactin is an F-actin binding protein and the substrate of numerous cellular kinases (reviewed in reference 25). Cortactin can weakly stimulate Arp2/3 directly or more potently via interaction with N-WASP. The activation of N-WASP is dependent on the SH3 domain of cortactin and is regulated by phosphorylation of serine and tyrosine residues in a proline-rich domain located upstream of the SH3 domain itself (25).

Given the propensity of cortactin to be present at sites of dynamic actin remodeling, it is not surprising that cortactin is detected in the actin-rich pedestals triggered by EHEC (7). Furthermore, its importance in these structures is underscored by the fact that truncated forms of cortactin have been reported to exert a dominant-negative effect on pedestal formation, and depletion of cortactin from HeLa cells by small interfering RNA treatment abolished pedestal formation (7). Recently, Cantarelli et al. have shown that cortactin can bind the N terminus of Tir and the proline-rich repeats of TccP (8). The fact that cortactin can activate N-WASP by itself, is able to bind Tir, and can interact with TccP led to the hypothesis that it might be the adaptor protein connecting Tir and TccP during infection (8). Alternatively, cortactin may contribute to an alternative pathway of actin assembly, such as the inefficient TccP-independent actin polymerization triggered by EHEC. The aim of this study was to investigate cortactin recruitment in the context of EHEC-triggered actin polymerization in vitro and A/E lesion formation ex vivo.

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Bacterial strains. The bacterial strains used in this study are listed in Table 1. The bacteria were grown in Luria-Bertani (LB) medium or in Dulbecco's modified Eagle's medium supplemented with kanamycin (50 µg·ml^–1) and/or ampicillin (100 µg·ml^–1) when necessary.

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TABLE 1. Bacterial strains and plasmids used in this study

Cell culture and transfection. HeLa, Swiss 3T3 (ATCC), and N-WASP^–/– mouse embryo fibroblast cell lines (kindly supplied by S. Snapper) were grown under conventional conditions. Cells were seeded on glass coverslips (13-mm diameter) in 24-well plates at a density of 5 x 10⁴ cells per well 48 h before infection. Where appropriate, the cells were transfected 24 h after being seeded using Jet-PEI-RGD (Polyplus) in accordance with the manufacturer's protocol.

Plasmids. The plasmids used in this study are listed in Table 1. For bacterial expression of tir, primers F1 (5'-ATGCCTATTGGTAATCTTGGTC-3') and R1 (5'-AGGCTGCAGTTAGACGAAACGATGGGATC-3') were used to amplify tir from wild-type EHEC genomic DNA strain TUV 93-0. PCR products were cloned between the SmaI and PstI restriction sites of pSA10 (23), a vector containing multiple cloning sites downstream of the tac promoter, generating plasmid pICC421. Amino acid substitutions were introduced into Tir_EHEC using the QuikChange II Site-Directed Mutagenesis kit (Stratagene), pICC421 as a template, and primers F2 (5'-AGGGACCGTGCAGAATCCGGCTGCTGATGTTAAAACATCG-3'), R2 (5'-CGATGTTTTAACATCAGCAGCCGGATTCTGCACGGTCCCT-3'), F3 (5'-TCCCAATGTGAATAATTCAATTGCTGCTGCAGCTGCATTAGCTTCACAAACCGACGG-3'), and R3 (5'-CCGTCGGTTTGTGAAGCTAATGCAGCTGCAGCAGCAATTGAATTATTCACATTGGGA-3') to generate Tir_Y458A and Tir_P17-23A derivatives, plasmids pICC422 and pICC423, respectively.

Infection and immunofluorescent staining. EHEC cultures, grown in LB medium for 8 h, were diluted 1:500 in Dulbecco's modified Eagle's medium and grown statically overnight at 37°C in a 5% CO₂ atmosphere prior to infection. The cells were infected for 5 h, washed three times in phosphate-buffered saline, and fixed for 20 min in 4% paraformaldehyde. The infected monolayers were permeabilized with 0.1% Triton for 4 min and labeled by indirect immunofluorescent staining. Goat polyclonal E. coli O157:H7 (Fitzgerald Industries International) and rabbit polyclonal Tir_EHEC (2) antisera were diluted 1:500. Mouse monoclonal anti-cortactin antibody and anti-hemagglutinin (HA) monoclonal antibody HA.11 (Convance) were diluted 1:200. Rhodamine- and Alexa 633-conjugated phalloidin (Invitrogen) were used at a dilution of 1:500; Oregon green-conjugated phalloidin (Invitrogen) was used at a dilution of 1:100. Donkey aminomethylcoumarin acetate-, Cy5-, rhodamine-, and Cy2-conjugated species-specific secondary antibodies (Jackson Immunoresearch Laboratories) were diluted 1:200. Coverslips were mounted with Pro-Long Gold antifade reagent (Invitrogen) and analyzed using a Zeiss Axioimager fluorescence microscope. Representative images of multiple experiments (typically three) were processed using axiovision software. Recruitment of cortactin and TccP-HA was quantified by counting a total of 4,400 adherent bacteria from two independent experiments.

Preparation of lysates for detection of exogenously expressed proteins by Western blotting. Whole-cell extracts were prepared by scraping transfected cell monolayers into protein-denaturing buffer and boiling them for 5 min prior to polyacrylamide gel electrophoresis and Western blotting. Primary antibodies were diluted 1:1,000 and detected using porcine anti-rabbit or anti-mouse immunoglobulin G (IgG) horseradish peroxidase-conjugated (Dako) secondary antibodies and ECLplus detection reagent (GE Healthcare).

Human in vitro organ cultures (IVOC) and immunofluorescence staining of cryosections. Pediatric tissue was obtained with fully informed parental consent and local ethical committee approval using grasp forceps during routine endoscopic investigation of intestinal disorders. Mucosal biopsy specimens from the terminal ileum that appeared macroscopically normal were taken for organ culture experiments as described previously (17). The biopsy specimens were infected with wild-type EHEC O157:H7, an isogenic tccP deletion mutant, or enteraggregative E. coli (EAEC) O42 for 8 h. An uninfected biopsy specimen was included in each experiment to exclude endogenous bacterial infection. Adherence was examined using tissue from four patients (between 74 and 198 months old) by cryosectioning and immunostaining as described previously (24). In each case, the comparison between different bacterial strains was made using samples from the same patient. For immunofluorescence, samples were embedded in OCT compound (Sakura), snap frozen in liquid nitrogen, and stored at –70°C until they were used. Serial sections 8 µm thick were cut with an MTE cryostat (SLEE Technik), picked up on poly-L-lysine-coated slides, and air dried. The tissue sections were fixed in formalin for 10 min and blocked with 0.5% bovine serum albumin, 2% normal goat serum in phosphate-buffered saline for 20 min at room temperature. The slides were incubated with rabbit polyclonal anti-Tir_EHEC or mouse monoclonal anti-cortactin for 60 min at room temperature, washed, and incubated in Alexa Fluor 647-conjugated goat anti-rabbit IgG or Alexa Fluor 488-conjugated goat anti-mouse IgG (Molecular Probes) for 30 min. Counterstaining of bacteria and cell nuclei was performed using propidium iodide (Sigma). The sections were analyzed with a Zeiss LSM 510 confocal laser scanning microscope.

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Recruitment of cortactin to the site of EHEC adhesion in vitro is dependent on Tir_Y458. Tir_Y458 has been implicated in actin polymerization and the recruitment of TccP (3, 4). In order to determine if Tir_Y458 plays a role in the recruitment of cortactin to sites of bacterial attachment, we infected Swiss 3T3 fibroblasts with wild-type EHEC, EHECtir, and EHECtir complemented with plasmids encoding either wild-type Tir (pTir_WT/pICC421) or Tir_Y458A (pTir_Y458A/pICC422). Immunofluorescent staining confirmed that Tir was clustered beneath adherent wild-type and EHECtir expressing either wild-type Tir or Tir_Y458A (Fig. 1). Actin polymerization and cortactin were detected only beneath adherent bacteria in cells infected with wild-type EHEC and EHECtir expressing wild-type Tir, but not with EHECtir expressing Tir_Y458A (Fig. 1). These results show that Y458 is essential for both actin polymerization and the recruitment of cortactin.

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FIG. 1. Recruitment of cortactin to the site of EHEC adhesion in vitro is dependent on TirY458 and TccP. Swiss 3T3 fibroblasts were infected with wild-type (WT) EHEC, EHECtir, EHECtccP, or EHECtir complemented with a plasmid encoding wild-type Tir or TirY458A. The bacteria were detected in UV light using a polyclonal goat anti-E. coli O157 EHEC antibody, and Tir was detected in far-red light using a rabbit polyclonal anti-TirEHEC antibody. Cortactin and actin were labeled green and red, respectively, using a mouse monoclonal anti-cortactin antibody and Rhodamine Red-X-conjugated phalloidin. Immunofluorescence showed Tir under all adherent EHEC strains except EHECtir. Actin polymerization and recruitment of cortactin was seen under attached wild-type EHEC and EHECtir bacteria complemented with a plasmid encoding wild-type Tir. Neither actin nor cortactin was detected beneath adherent EHECtir, EHECtir complemented with a plasmid encoding TirY458A,or EHECtccP bacteria. Shown are monochrome images of the UV, green, red, and far-red fluorescent channels and a merged color image.

Cortactin is recruited to the site of EHEC adhesion in vitro downstream of TccP. In order to determine if overexpression of cortactin can enhance the inefficient Tir_EHEC-dependent and TccP-independent actin polymerization, HeLa cells were transiently transfected with expression vectors encoding green fluorescent protein (GFP)-cortactin or GFP as a control. Expression of both constructs was confirmed by Western blotting (Fig. 2A). GFP- and GFP-cortactin-expressing cells were infected for 5 h with EHECtccP (strain ICC185), EHECtccP complemented with an expression vector encoding HA-tagged TccP (pICC369), and wild-type EHEC as a control. Fixed monolayers were processed for immunofluorescent staining of bacteria and Tir. Microscopic analysis revealed that Tir was concentrated beneath wild-type EHEC, EHECtccP, and complemented EHECtccP expressing HA-tagged TccP (Fig. 2B), indicating that overexpression of GFP-cortactin did not affect Tir recruitment underneath adherent bacteria. Furthermore, staining of bacteria and F-actin revealed that only wild-type EHEC and EHECtccP complemented with HA-tagged TccP recruited GFP-cortactin and triggered actin pedestal formation. No significant GFP signal was seen in the pedestals triggered by these bacteria on cells expressing GFP (Fig. 2C). Wild-type EHEC and EHECtccP expressing HA-tagged TccP triggered actin polymerization with similar efficiencies on cells expressing GFP and GFP-cortactin. As expected, EHECtccP was unable to efficiently trigger cortactin recruitment or actin pedestal formation on untransfected cells (Fig. 1) or cells transfected with GFP (Fig. 2C). Moreover, EHECtccP was not able to trigger formation of actin pedestals or recruit cortactin in GFP-cortactin-overexpressing cells (Fig. 2C). Together with the data shown in Fig. 1, these results show that overexpression of cortactin does not overcome the deficiency of EHECtccP in triggering efficient actin polymerization and that recruitment of cortactin to the site of EHEC attachment occurs downstream of TccP recruitment.

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FIG. 2. Cortactin recruitment in vitro is dependent on TccP. (A) Lysates of transfected HeLa cells were immunoblotted for expression of GFP or GFP-cortactin. The migratory position of GFP is indicated with an asterisk, and GFP-cortactin is indicated with an arrowhead. Tubulin immunoblotting showed that equivalent protein levels were present within HeLa lysates. The positions of molecular mass standards are shown on the left. (B) Expression of GFP-cortactin in HeLa cells does not impair Tir recruitment under adherent EHEC. GFP-cortactin-transfected HeLa cells were infected with wild-type EHEC, EHECtccP (ICC185), and EHECtccP expressing HA-tagged TccP from a plasmid (pICC369). The infected cells were stained with goat anti-E. coli O157 EHEC to label bacteria (blue) and rabbit polyclonal anti-TirEHEC antibody (red). (C) GFP- and GFP-cortactin-transfected HeLa cells were infected with wild-type (WT) EHEC, EHECtccP, and EHECtccP expressing TccP-HA. The bacteria were detected in UV light using a polyclonal goat anti-E. coli O157 EHEC antibody, and F-actin was stained using Rhodamine Red-X-conjugated phalloidin. GFP-cortactin, colocalizing with actin, was detected beneath adherent wild-type EHEC and EHECtccP bacteria expressing TccP-HA, but not under EHECtccP bacteria. Shown are monochrome images of the UV, GFP, and red fluorescent channels. Scale bar, 2 µm.

Cortactin is recruited to the site of EHEC adhesion in vitro downstream of N-WASP. TccP is responsible for the recruitment of N-WASP during in vitro EHEC infection (6, 13). In order to determine if N-WASP is implicated in the recruitment of cortactin, we infected an N-WASP-deficient fibroblast cell line (N-WASP^–/– mouse embryo fibroblasts), transfected with either GFP-tagged N-WASP or GFP alone as a control, with EHECtccP (ICC185) expressing HA-tagged TccP from a plasmid (pICC369). Western blotting of lysates of transfected cells confirmed the expression of the two constructs and that N-WASP expression did not alter cortactin levels in the cell (Fig. 3A).

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FIG. 3. Recruitment of TccP and cortactin during EHEC infection of an N-WASP-deficient cell line. (A) Lysates of transfected N-WASP–/– mouse embryo fibroblasts were immunoblotted for GFP or GFP-tagged N-WASP using anti-GFP (left) and anti-N-WASP (right) antibodies. The migratory position of GFP is indicated with an asterisk, and GFP-N-WASP is indicated with an arrowhead. Overexpression of N-WASP did not affect cortactin levels. The positions of molecular mass standards are shown. (B) TccP, but not cortactin or F-actin, is efficiently recruited to sites of EHEC adherence in the absence of N-WASP. GFP- or GFP-N-WASP-transfected N-WASP–/– cells were infected with EHECtccP (ICC185) expressing TccP-HA (pICC369). The infected cells were treated with anti-HA antibody to visualize, in red, epitope-tagged TccP (left); with mouse monoclonal anti-cortactin antibody (middle); and with phalloidin (right) to detect F-actin. The bacteria were labeled in UV light (pseudocolor, blue) using a goat polyclonal anti-E. coli O157 antibody.

N-WASP^–/– cells were transfected with GFP, infected with EHECtccP expressing HA-tagged TccP, and immunostained with anti-HA and anti-cortactin antibodies. TccP-HA was found under 90% ± 6% of adherent bacteria, and weak cortactin staining was seen under 9.5% ± 2% of attached EHEC bacteria (Fig. 3B, left and center, respectively). In contrast, infection of N-WASP^–/– cells transfected with GFP-N-WASP restored intense cortactin recruitment beneath 95% ± 0% of adherent EHECtccP bacteria expressing HA-tagged TccP. Anti-HA staining confirmed that, similarly to cells transfected with GFP, TccP-HA was recruited beneath 92% ± 7% of adherent bacteria in cells transfected with GFP-N-WASP. As expected, no HA staining was detected beneath EHECtccP or wild-type EHEC (not shown) bacteria, nor were any of the strains tested able to trigger the formation of distinct actin pedestals on N-WASP^–/– cells transfected with GFP (Fig. 3B, right). Taken together, these data show that while TccP is recruited to Tir in the absence of N-WASP, recruitment of cortactin is N-WASP dependent. These results suggest that cortactin is not involved in linking Tir and TccP during infection of cultured cells in vitro.

The N terminus of Tir is dispensable for cortactin recruitment. In vitro, cortactin, via its SH3 domain, reportedly binds directly to the N terminus of Tir (8), which contains a polyproline region (amino acids 17 to 23) that is a putative SH3 domain-binding site. We therefore investigated if the N terminus of Tir and the polyproline region contained within it have a role in cortactin recruitment under adherent bacteria. To this end, we infected Swiss 3T3 fibroblasts with EHECtir complemented with a plasmid encoding Tir in which all five N-terminal proline residues were replaced with alanine, Tir_P17-23A (pTir_P17-23A/pICC423). In parallel, full-length HA-Tir and a truncated HA-Tir5-221 derivative (4) lacking the entire N terminus were expressed in HeLa cells via transfection prior to infection with EHECtir. Phalloidin staining revealed that translocated Tir_P17-23A (Fig. 4A) and ectopically expressed HA-Tir and HA-Tir5-221 (Fig. 4B) all complemented the ability of EHECtir to induce actin-rich pedestals; in all cases, Tir was located at the tip of the pedestal (Fig. 4). Staining with an anti-cortactin antibody revealed, similarly, that cortactin was recruited beneath adherent EHECtir bacteria expressing Tir_P17-23A on fibroblasts (Fig. 4A) and EHECtir bacteria following infection of HA-Tir5-221-transfected cells, but not on mock-transfected cells (Fig. 4B). Taken together, these data indicate that the Tir polyproline region and, more generally, the whole N terminus of Tir are not necessary for efficient actin pedestal formation and cortactin recruitment.

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FIG. 4. The N terminus of Tir is dispensable for recruitment of cortactin. (A) Swiss 3T3 fibroblasts were infected with EHECtir complemented with a plasmid encoding TirP17-23A. Staining as for Fig. 1 revealed actin polymerization and recruitment of cortactin under attached bacteria. (B) Mock-, HA-Tir-, or HA-Tir5-221-transfected HeLa cells were infected with EHECtir. The infected cells were treated with polyclonal goat anti-E. coli O157 antibody to label bacteria (blue), with Oregon green phalloidin to detect F-actin (green), and with either a mouse monoclonal anti-HA antibody (left) to visualize epitope-tagged Tir (red) or a mouse monoclonal anti-cortactin antibody (right) (red). Shown are separate monochrome images of the UV, red, and green fluorescence channels and a merged color image.

Cortactin is recruited to the ex vivo site of EHEC adhesion independently of TccP. We have recently shown, using ex vivo and in vivo infection models, that unlike the induction of actin polymerization in vitro, TccP is dispensable for A/E lesion formation on mucosal surfaces (13, 22, 26). We therefore studied cortactin recruitment to the site of EHEC adhesion during infection of human IVOC. To this end, intestinal biopsy specimens from the terminal ileum were infected with wild-type EHEC and EHECtccP (ICC185). An uninfected biopsy specimen was used as a negative control. Immunofluorescent staining of cryosections for cortactin showed general cytoplasmic and membrane staining of enterocytes in uninfected control samples (Fig. 5). In contrast, intense cortactin staining was detected under every adherent wild-type EHEC and EHECtccP bacterium (30/30) (Fig. 5), indicating that cortactin recruitment on mucosal surfaces is independent of TccP. As EHECtir does not bind IVOC, we could not use this strain as a control. Instead, we employed EAEC O42, which binds to terminal ileal mucosa (21) by a Tir-independent mechanism. No cortactin was detected at the site of any attached EAEC bacterium (10/10) (Fig. 5), suggesting that recruitment of cortactin to EHEC O157:H7 is specific.

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FIG. 5. Immunofluorescence staining of cryosections of human terminal ileum infected with wild-type EHEC, EHECtccP, or EAEC. Whereas general cytoplasmic and membrane cortactin staining could be observed in enterocytes of noninfected and EAEC-infected samples, intense cortactin staining beneath Tir-expressing EHEC bacteria and an isogenic tccP deletion mutant was evident (upper row; green in merged images). Sections were also stained for Tir (blue in merged images) and counterstained with propidium iodide (red) to visualize bacteria and cell nuclei. Cortactin (green) and Tir (blue) staining overlap in the merged image.

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A number of pathogenic bacteria target cortactin during infection. For example, Listeria monocytogenes and Shigella flexneri exploit cortactin for invasion of mammalian host cells (reviewed in reference 25). In addition, cortactin is reported to bind the N terminus of Tir (8) and to play a role in triggering actin polymerization at the site of EHEC adhesion to eukaryotic cells (7). Here, we have studied the recruitment of cortactin during EHEC infection of cultured cells and human IVOC.

We have shown that overexpression of cortactin does not enhance actin polymerization after infection of cultured cells with EHECtccP, suggesting that cortactin is unlikely to contribute to the inefficient TccP-independent actin polymerization pathway triggered by Tir_EHEC in vitro. We have shown that although wild-type Tir is focused under attached EHECtccP bacteria and Tir_Y458A is focused under EHECtir(pTir_Y458A) bacteria (which express TccP but are unable to recruit it under attached bacteria), this does not lead to recruitment of cortactin. These results suggest Tir does not directly recruit cortactin, but rather, cortactin recruitment to the site of bacterial attachment is TccP dependent. Moreover, cortactin was recruited to EHECtir expressing Tir_P17-23A and to EHECtir after infection of cells transfected to express Tir5-221. These results show that despite the reported ability of cortactin to bind the N terminus of Tir directly (8), this binding activity is not sufficient for recruitment of cortactin to the site of intimate bacterial attachment or for actin polymerization.

Cortactin is able to bind to both Tir and TccP in vitro (8), thus implicating cortactin as a putative host adaptor linking the two bacterial effectors required for actin pedestal formation in vitro. To test this hypothesis experimentally, we infected N-WASP-deficient cells complemented with either GFP-N-WASP or GFP alone. We established that TccP is recruited to Tir in the absence of N-WASP and then reasoned that if cortactin was the adaptor, it would also be recruited to Tir in the absence of N-WASP. However, cortactin was recruited only in cells transfected with GFP-N-WASP. Together, these results indicate that in vitro, cortactin is recruited to the site of EHEC adhesion, not directly by Tir but downstream of TccP and N-WASP. Although not involved in linking Tir and TccP, cortactin might have a role in stabilizing the actin polymerization complex, as inferred by Cantarelli et al. (8).

It has recently been shown that TccP is not needed for colonization of mucosal surfaces ex vivo (1) and in vivo (22, 26). In order to demonstrate if the data we gathered for cortactin in vitro could be extended to the infection of mucosal surfaces, we infected human terminal ileal biopsy specimens with wild-type EHEC O157:H7, EHECtccP, and EAEC O42 as a control. In contrast to the recruitment of cortactin in vitro, which is TccP dependent, we found that cortactin recruitment to the site of EHEC attachment in human IVOC was TccP independent.

Previous data obtained from analysis of EHEC-infected intestinal tissue using transmission electron microscopy revealed that regions of electron-dense staining characteristic of localized actin assembly were often not associated with intimately attached EHECtccP bacteria (22) or an analogous tccP-minus EPEC strain expressing EHEC O157-like Tir (1). These observations suggest that EHECtccP has a diminished capacity to generate actin pedestals (but not A/E lesions) in vivo, despite retaining the ability to recruit cortactin. Importantly, the concentration of cortactin at the sites of EHEC A/E lesions appears to be pathogen specific, as the protein was not recruited to sites of EAEC adhesion. One explanation for these data is that A/E lesions induced in ex vivo intestinal epithelial cells are not strictly equivalent to actin polymerization in cultured cells in vitro, both in terms of the signaling pathways (10) and in the host cell proteins that accumulate therein. Cortactin, as it is recruited to sites of bacterial adhesion ex vivo via a TccP- and N-WASP-independent pathway, might have a role during EHEC infection of mucosal surfaces that is more influential than that of cultured cells in vitro, and its role in mediating A/E lesion formation is currently under investigation.

 
We thank V. Koronakis (Cambridge University, United Kingdom) for kindly supplying anti-N-WASP antiserum, Scott Snapper of Harvard Medical School for the N-WASP knockout and control mouse embryo fibroblast cell lines, S. Lommel (Institute for Cell Biology, University of Bonn, Bonn, Germany) for the GFP-N-WASP, and L. Machesky (Beatson Institute, Glasgow, United Kingdom) for the cortactin expression vectors. Monoclonal anti-tubulin antibody developed by M. Klymkowsky was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the Department of Biological Sciences, University of Iowa, Iowa City, IA 52242.

J.M.L. is supported by NIH R01-AI46454. A.D.W. is funded by a BBSRC studentship. Work in the laboratory of A.D.P. was supported by the NIH (grant R37AI21657 to J. B. Kaper). This project was supported by the Wellcome Trust.

 
* Corresponding author. Mailing address: Division of Molecular and Cellular Biology, Flowers Building, Imperial College London, London SW7 2AZ, United Kingdom. Phone: 44 020 2594 5253. Fax: 44 020 5794 3069. E-mail: g.frankel{at}imperial.ac.uk

Published ahead of print on 4 August 2008.

Editor: J. B. Bliska

A.M. and A.D.W. contributed equally to this work.

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Infection and Immunity, October 2008, p. 4669-4676, Vol. 76, No. 10
0019-9567/08/$08.00+0     doi:10.1128/IAI.00140-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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