Repetitive N-WASP–Binding Elements of the Enterohemorrhagic Escherichia coli Effector EspF_U Synergistically Activate Actin Assembly

Intimin-mediated clustering of Tir and EspF_U is sufficient to promote actin pedestal formation

During infection, EHEC is capable of translocating more than 30 effector proteins into the host cell [9]. At least two of these effectors, EspF_U and EspF, are known to directly activate N-WASP [28]–[29],[33], while additional proteins stimulate signaling pathways that may also lead to N-WASP-mediated actin polymerization [34]. However, only two effectors, Tir and EspF_U, have been shown to be crucial for EHEC pedestal formation in genetic deletion studies. Moreover, KC12, an EPEC strain in which EPEC tir has been replaced by EHEC tir, is defective at actin pedestal formation, whereas expression of EspF_U by KC12 allows pedestal formation at high efficiency in manner indistinguishable from that of EHEC [28]. These results are consistent with the possibility that Tir and EspF_U are the only effectors essential for EHEC-mediated actin pedestal formation. To definitively test whether EspF_U is the only effector in addition to Tir that is required for actin pedestal assembly, mammalian cells were transfected with plasmids encoding derivatives of EHEC Tir and EspF_U in the absence of all other bacterial factors. Tir was N-terminally tagged with an HA epitope, and its first transmembrane domain replaced with the transmembrane segment of the Newcastle Disease Virus HN surface protein to promote efficient plasma membrane localization [31]. EspF_U was fused to GFP at its N-terminus and a 5myc tag at its C-terminus to allow detection by fluorescence microscopy and immunoblotting, respectively (Figure 1A). In addition to full-length EspF_U, we also generated GFP-fusions carrying only its N-terminal 88 residues (EspF_U-N) or its C-terminal repeats (EspF_U-R1-6) (Figure 1B). To cluster Tir in the plasma membrane, transfected cells were treated with a non-pathogenic strain of E. coli that was engineered to express intimin (Figure 1A), and binds selectively to Tir-expressing cells [31].

To evaluate actin pedestal formation on cells additionally co-expressing EspF_U, only those cells exhibiting GFP fluorescence were examined. Adherent bacteria were identified by DAPI-staining and F-actin was visualized using fluorescent phalloidin. Bacteria that bound to cells co-expressing Tir and full-length EspF_U were associated with robust localized actin assembly, indicating that clustering of Tir in the presence of EspF_U is sufficient to trigger pedestal formation (Figure 1C). In addition, pedestals were formed on cells expressing the C-terminus of EspF_U, but not cells expressing the N-terminus, indicating that the activity of EspF_U in pedestal formation resides entirely within the repeat region.

Experimental clustering of a single EspF_U repeat bypasses the requirement for the Tir C-terminus during actin pedestal formation

While deletion of the N-terminal cytoplasmic domain of Tir has a modest effect on actin assembly [31], the C-terminus of EHEC Tir contains a tripeptide sequence that is critical for both recruitment of EspF_U and pedestal formation [30]. Given that Tir and EspF_U do not appear to bind one another directly [28]–[32], and that EspF_U can activate N-WASP, the simplest model for pedestal formation is that the C-terminus of Tir serves to recruit a host factor that mediates the Tir-EspF_U interaction, thereby indirectly clustering EspF_U beneath the plasma membrane. If the major role of such a host protein is to act as an adaptor between Tir and EspF_U, then artificial clustering of EspF_U at the plasma membrane should bypass the requirement for the C-terminus of Tir during pedestal formation. To test this possibility, we replaced the C-terminal cytoplasmic domain of HN-Tir with the C-terminal repeats of EspF_U, expressed this Tir-EspF_U fusion in mammalian cells, and clustered it at the plasma membrane using antibodies directed against the extracellular region of Tir and formalin-fixed Staphylococcus aureus particles to engage Tir-bound antibodies (Figure 2A). We found that clustering of HN-Tir-EspF_U-R1-6, which encompasses all 6 repeats, resulted in high levels of actin pedestal formation. Measurement of the fraction of cells harboring five or more pedestals yielded a pedestal “index” of nearly 90% (Figure 2B and 2C). As expected, the control HN-Tir-EspF_U-N fusion did not trigger significant actin assembly.

The number of repeats present in the C-terminus of EspF_U from different EHEC isolates varies from two to eight, and an espF_U mutant of the prototype strain EDL933 could not be complemented for pedestal formation by a plasmid encoding a truncation with only one repeat [32],[35]. To determine whether the quantity of repeats affects the efficiency of pedestal formation, we assessed actin assembly upon clustering of Tir-EspF_U chimeras containing variable numbers of EspFu repeats. All of the derivatives, including HN-Tir-EspF_U-R1, a fusion containing only a single repeat, were capable of stimulating actin assembly (Figure 2B and 2C). Quantitation of the pedestal index revealed that HN-Tir-EspF_U-R1 was roughly half as efficient at generating pedestals as the fusions containing multiple repeats (Figure 2C). These results suggest a remarkably simple model in which the central function of Tir is to promote clustering of the peptide repeats of EspF_U beneath the plasma membrane. In fact, clustering of a single repeat is sufficient to trigger actin pedestal formation.

Multiple EspF_U repeats are required for full pedestal formation activity when expressed in the presence of full-length Tir

We next sought to further investigate the activity of EspF_U derivatives harboring varying numbers of repeats in experimental systems involving full-length Tir instead of Tir-EspF_U chimeras. To avoid potential variability in the efficiency of type III translocation of different EspF_U derivatives, we expressed GFP-EspF_U truncations in mammalian cells, and tested their ability to complement KC12, the equivalent of an EHECΔespF_U mutant [28], for actin pedestal formation. As expected [36], KC12 generated pedestals on cells expressing GFP-EspF_U-R1-6, which carries the entire repeat region (Figure 3A). Moreover, all of the EspF_U derivatives that contained at least two repeats were also capable of complementing KC12 for pedestal formation. However, KC12 did not generate pedestals efficiently on GFP-EspF_U-R1-expressing cells. Only 1–4 pedestals were occasionally observed on cells transfected with this variant (data not shown). This discrepancy in actin assembly cannot be attributed to differences in protein levels, because immunoblotting revealed that GFP-EspF_U-R1 was well expressed in mammalian cells (data not shown).

To independently assess pedestal formation in the absence of other E. coli effectors that might compete with Tir or EspF_U for host target proteins, we also utilized the co-transfection approach described in Figure 1. HN-Tir along with GFP-EspF_U fusions carrying different numbers of repeats were expressed in mammalian cells and clustered by treating cells with intimin-expressing bacteria. When Tir was co-expressed with EspF_U constructs harboring multiple repeats, intensely staining F-actin pedestals were formed in high numbers (Figure 3B). On cells expressing HN-Tir and GFP-EspF_U-R1, few bacteria were associated with intense pedestals, whereas most were associated with weakly staining F-actin (arrowheads). These results indicate that although EspF_U derivatives containing a single repeat can function for pedestal formation after clustering of full-length Tir in mammalian cells, the presence of 2–6 repeats is required for robust actin assembly.

Multiple EspF_U repeats are required for efficient binding of native N-WASP in mammalian brain extract

Since actin pedestal formation varied depending on the number of EspF_U repeats, we next examined whether the efficiency of pedestal formation correlates with the ability of the different EspF_U truncations to interact with N-WASP. To characterize the interaction between N-WASP and derivatives of EspF_U harboring different numbers of repeats in a complex environment reflective of the mammalian cytosol, we generated protein extracts from pig brains, which are rich in actin cytoskeleton-associated factors, including N-WASP, WIP, and the Arp2/3 complex (data not shown). We also purified recombinant N-terminally His10- and C-terminally 5myc-tagged EspF_U derivatives consisting of 1 to 6 repeats, or as a negative control, an N-terminal fragment of EspF_U (Figure 4A). Magnetic beads saturated with these EspF_U truncations were then incubated with brain extracts, and N-WASP that bound to the beads was detected by immunoblotting. EspF_U-R1-6, the derivative containing the entire C-terminus of EspF_U, pulled down N-WASP, whereas the EspF_U-N control fragment did not (Figure 4B). The efficiency of N-WASP pulldown appeared to be slightly lower for variants containing fewer than four repeats, whereas mutants containing only a single repeat bound N-WASP at substantially decreased levels (R1-6 = R1-4>R1-3 = R1-2≫R1 = R6).

Since it is possible that saturating beads with a single EspF_U repeat may artificially mimic derivatives with multiple repeats due to the density of protein clustered on the beads, we also assessed the interaction between EspF_U and native N-WASP under conditions where EspF_U concentrations could be more stringently controlled. Therefore, His-EspF_U derivatives were first added to extract at a defined concentration (1 µM), and then collected using cobalt-chelated magnetic particles. Immunoblotting revealed that N-WASP interacted efficiently with EspF_U derivatives containing 6 or 4 repeats (Figure 4C). Slightly lower levels of N-WASP bound to EspF_U proteins harboring 3 or 2 repeats, and constructs containing a single repeat unit pulled-down N-WASP at barely-detectable levels (R1-6 = R1-4>R1-3>R1-2>>>R1 = R6). Interestingly, increasing the concentration of EspF_U truncations had little effect on the quantity of N-WASP pulled-down by those proteins. Thus, although one repeat is capable of binding N-WASP in brain extracts, stepwise increases in binding are associated with the presence of additional EspF_U repeats within the same molecule, with an apparent plateau at 4–6 repeats.

EspF_U repeats cause titratable increases in actin assembly in brain extract

EspF_U derivatives containing the N-terminal secretion domain and two or more repeats have been shown to stimulate N-WASP-Arp2/3–mediated actin assembly using purified proteins in vitro, while a truncation containing a single repeat barely triggered assembly [32]. To similarly assess the ability of EspF_U to promote actin polymerization, but in the presence of native components, we examined the ability of recombinant EspF_U variants to stimulate actin polymerization in brain extract. Purified EspF_U derivatives were added to extract supplemented with pyrene-labeled actin, and polymerization kinetics were measured fluorometrically [37]. Consistent with analyses of pedestal formation in cells, EspF_U-R1-6 accelerated actin assembly in extract, while EspF_U-N did not (Figure 5A). The degree of stimulation by EspF_U-R1-6 was dose dependent, and peaked at 20 nM (Figure 5B), although polymerization rates were slower than those typically observed in a completely purified experimental system, presumably due to the presence of inhibitory factors in the extract. Concentrations above 20 nM did not increase the rate of assembly, and levels greater than 50 nM actually resulted in slower kinetics (not shown), implying that polymerization in extract is refractory in the presence of high levels of EspF_U.

To characterize the relationship between the number of EspF_U repeats and the ability to stimulate actin assembly, pyrene-actin polymerization was also measured when the recombinant EspF_U truncations were added to extract. When tested at 20 nM, the optimal concentration for actin assembly mediated by the EspF_U derivative containing all 6 repeats, EspF_U variants containing 2, 3, or 4 repeats accelerated actin assembly, and the rate of polymerization positively correlated with the number of repeats that were present (Figure 5C). In contrast, neither EspF_U-R1 nor EspF_U-R6, the truncations containing a single repeat, caused a significant acceleration of actin assembly in this assay system. Notably, the kinetics of actin assembly in these studies correlated with the ability of the EspF_U constructs to interact with N-WASP in the extract (Figure 4). Consistent with such results, increasing the concentration of EspF_U variants harboring less than 3 repeats did not enhance actin assembly (data not shown). Collectively, these data indicate that the presence of multiple repeats within the same polypeptide is important for triggering actin polymerization in an extract system designed to mimic a complex native environment.

EspF_U repeats synergistically activate N-WASP/WIP–mediated actin assembly in vitro

Under physiological conditions, N-WASP stably associates with WIP or it homologs CR16 and WIRE/WICH [21],[38]. In addition, WIP is capable of inhibiting Cdc42-mediated activation of N-WASP [20] and, unlike recombinant N-WASP, the native N-WASP/WIP complex is insensitive to Cdc42 treatment [21], suggesting that regulation of N-WASP in isolation may not accurately reflect physiological N-WASP regulation in vivo. We therefore examined whether EspF_U can trigger Arp2/3-mediated actin assembly in the presence of an N-WASP/WIP complex. We purified a recombinant complex containing N-WASP and WIP at 1∶1 stoichiometry and also contained a trace amount of insect cell actin (see Materials and Methods; Figure 6A). We also purified recombinant Arp2/3 complex [39], and examined the basal activity of the N-WASP/WIP complex using the pyrene-actin polymerization assay. Consistent with the predicted autoregulation of N-WASP, the recombinant N-WASP/WIP complex caused only a small increase in actin assembly kinetics when compared to Arp2/3 alone (Figure 6B).

To determine if EspF_U could trigger actin assembly in this purified system, pyrene-actin polymerization was measured in the presence of recombinant EspF_U-R1-6, N-WASP/WIP, and Arp2/3 complex. Consistent with analyses of pedestal formation in cells and actin assembly in extracts, EspF_U-R1-6 accelerated actin assembly in this reconstituted system (Figure 6B). In the presence of 20 nM N-WASP/WIP and 20 nM Arp2/3, activation appeared to saturate at approximately 35 nM EspF_U-R1-6.

We next examined the abilities of the EspF_U truncations to stimulate actin assembly. By identifying the length of time each reaction took to reach half of the maximal F-actin concentration and measuring the rates of actin polymerization at these times, we directly compared the activity of each construct (Figure 6C). These experiments revealed that EspF_U-R1-6 and EspF_U-R1-4 were nearly indistinguishable at stimulating actin assembly, as the concentrations of these proteins that were required for reaching half of the maximal polymerization rate were approximately 4.0 nM and 5.7 nM, respectively. EspF_U-R1-3 was less active than either the 6- or 4-repeat constructs at all concentrations tested, and it stimulated half-maximal assembly at a concentration of 19.4 nM. EspF_U-R1-2 was substantially less active than the 3-repeat derivative (half-maximal at 78 nM), while the single repeat constructs EspF_U-R1 and EspF_U-R6 were even less active (half-maximal at 99–117 nM). Even when present at very high concentrations (>500 nM), the EspF_U derivatives harboring 1–2 repeats could not accelerate polymerization to rates comparable to those elicited by 35 nM EspF_U-R1-6 (data not shown). Thus, in the presence of purified N-WASP/WIP and Arp2/3, the number of EspF_U repeats positively correlates with the rate of actin assembly.

To compare the activity of the EspF_U derivatives on the basis of repeat numbers, we normalized protein concentrations to the quantity of repeat units within each polypeptide. For example, 4.17 nM of the 6-repeat construct was considered equivalent to 8.33 nM of a 3-repeat truncation and 25 nM of a single repeat. Scaling of the data in this manner revealed that EspF_U derivatives containing greater numbers of repeats within the same protein had substantially higher activity than smaller constructs (Figure 6D). For example, when protein concentrations were normalized to 25 nM of repeats, the 6-repeat protein was actually twice as active as the 3-repeat protein, which was more than twice as active as the 2-repeat protein, which was roughly twice as active a 1-repeat protein. A similar trend was apparent at higher concentrations (50–200 nM), as longer EspF_U constructs always had greater levels of activity than smaller derivatives containing equimolar amounts of repeats. Hence, EspF_U repeats cooperate when present within the same protein to promote synergistic activation of N-WASP/WIP and Arp2/3-mediated actin assembly.

The GBD of N-WASP is a dominant negative inhibitor of EHEC pedestal formation and binds with high efficiency to a single EspF_U repeat in yeast two-hybrid assays

Insight into the ability of multi-repeat EspF_U derivatives to pulldown N-WASP from brain extracts and stimulate N-WASP/WIP-mediated actin assembly in vitro would be enhanced by a better understanding of EspF_U binding by N-WASP, which is mediated by the GBD [28]–[29]. To further test the functional significance of this interaction during pedestal formation, we transfected mammalian cells with constructs expressing Flag-tagged N-WASP variants, each encompassing a different combination of N-WASP domains (Figure 7A), and then examined pedestal formation upon infection with EHEC. Consistent with previous observations showing that N-WASP residues 226–274 within the GBD mediate recruitment of N-WASP to sites of EHEC attachment [14], all tagged N-WASP derivatives containing the GBD (residues 151–273) were recruited to sites of bacterial adherence (Figure 7B, top row). In addition, quantitation of pedestal formation on transfected cells revealed that neither the N-terminal WH1 domain nor full-length N-WASP had a substantial effect on actin polymerization, whereas overexpression of each of the 4 GBD-containing derivatives that lacked the VCA domain, which is necessary for Arp2/3 activation, effectively abolished pedestal formation (Figure 7B, bottom row). This inhibition was specific, because overexpression of the GBD did not affect actin assembly triggered by an EPEC strain that generates pedestals independently of EspF_U (Figure 7C). As predicted by mapping of requirements for N-WASP recruitment to EHEC [14], an H208D point mutation that abrogates N-WASP binding by the GTPase Cdc42 had no effect on the inhibitory activity of the GBD. These results indicate that the interaction between EspF_U and the C-terminal portion of the N-WASP GBD is important for pedestal formation in mammalian cells.

Next, to begin to assess whether the positive correlation between the number of repeats within an EspF_U derivative and its ability to activate actin assembly is reflected in binding to the N-WASP GBD, we examined this interaction in yeast two-hybrid assays. As demonstrated previously [28], the N-WASP GBD interacted with the C-terminus of EspF_U that contains the repeat sequences, but not the EspF_U N-terminus (Figure 7D). In addition, all of the C-terminal subfragments of EspF_U, even those containing only a single repeat, interacted with the GBD in these assays. However, the degree of interaction did not positively correlate with increasing numbers of repeats. Rather, the number of repeats in EspF_U inversely correlated with the degree of expression of the lacZ reporter. Although reporter activity in the yeast two-hybrid assay reflects a number of parameters, including levels of expression and nuclear import of the binding partners, these results provide no evidence that the more efficient activation by multi-repeat EspF_U derivatives is a consequence of cooperativity in GBD binding.

Increasing the number of EspF_U repeats does not alter affinity for the GBD, but promotes the formation of an Arp2/3-containing complex

To further assess potential cooperativity in GBD binding by EspF_U, we examined the interactions of EspF_U derivatives with the GBD of WASP, the hematopoetic-specific homologue of N-WASP that has been shown to also promote actin pedestal formation in cells [14]. Recombinant EspF_U proteins containing 5, 2, or 1 repeats (R′1-5, R′4-5, and R′5, respectively) were generated with N- and C-terminal repeat boundaries based on recent structural studies that defined the GBD-binding sequences of EspF_U [40]–[42]. The affinity of the WASP GBD for these EspF_U constructs was determined using isothermal titration calorimetry and yielded predicted differences in stoichiometry (i.e., 5.7, 1.8, and 1.0 for the 5, 2, and 1 repeat derivatives, respectively) but similar dissociation constants (i.e., 83 nM, 95 nM, and 89 nM, respectively) (Figure 8A). Thus, variation in the number of EspF_U repeats is not associated with differences in affinity for the GBD.

We next tested whether oligomerization of N-WASP by adjacent EspF_U repeats instead might promote binding of the Arp2/3 complex. N-WASP_C, a C-terminal fragment of N-WASP that contains the GBD, PRD, and VCA domain, is a fragment previously shown to function in pedestal formation [14]. To determine whether EspF_U derivatives differing in repeat number also differed in their ability to form a complex containing N-WASP_C and Arp2/3, EspF_U variants containing 1 or 2 repeats, fluorescently labeled at their N-termini with AlexaFluor647, were incubated with equivalent molar concentrations of N-WASP_C in the absence or presence of the Arp2/3 complex. The relative size of EspF_U-containing complexes, detected by absorbance at 650 nM, was then determined by gel filtration chromatography. The single EspF_U repeat bound N-WASP at essentially stoichiometric levels when mixed in equimolar (2 µM) quantities, as determined by an increase in the apparent size (i.e., earlier elution profile) of EspF_U (Figure 8B left, red vs. blue peaks). The addition of 2 µM (i.e., a two-fold higher molar concentration) Arp2/3 caused ∼60% of the EspF_U to shift to an even more rapidly eluting fraction indicative of an EspF_U-N-WASP-Arp2/3 complex (Figure 8B, yellow profile). Very little of this complex was detected using 1 µM Arp2/3 (Figure 8B left, small shoulder in gray profile).

When 1 µM N-WASP_C was incubated with 0.5 µM of the 2-repeat EspF_U derivative (i.e., containing a normalized repeat concentration), the two proteins bound one another at essentially stoichiometric levels (Figure 8B, right, compare light blue and green profiles). However, in contrast to the requirement for 2 µM Arp2/3 to observe appreciable complex formation with a single repeat, the addition of as little as 0.5 µM Arp2/3 to N-WASP_C and the two repeat derivative resulted in the formation of an Arp2/3-containing complex (Figure 8B right, blue or pink profiles, respectively). The greater complex formation even at lower Arp2/3 concentration indicates that the ability of tandem EspF_U repeats to assemble N-WASP dimers (or by implication higher order multimers) may facilitate binding of the Arp2/3 complex, providing a likely source of inter-repeat cooperativity.

Since the assay demonstrating inter-repeat cooperation of EspF_U for the formation of an Arp2/3-containing complex described above involved the addition of only EspF_U, N-WASP_C, and Arp2/3, N-WASP-associated proteins such as WIP should not be required for cooperativity between the EspF_U repeats in actin assembly. Furthermore, domains of WASP (or N-WASP) other than the GBD (which binds EspF_U) and VCA (which binds Arp2/3) should also be dispensable. We therefore tested whether the EspF_U repeats were capable of synergistically activating a minimized (and normally autoinhibited [40]) derivative of WASP containing only the GBD and VCA domain. Similar to results using EspF_U truncations and an N-WASP/WIP complex, pyrene actin assays using WASP GBD-VCA revealed that the number of EspF_U repeats correlated with increased actin polymerization rates, even when protein concentrations were normalized to repeat units (Figure 8C). These results indicate that synergy in actin assembly require no WASP sequences other than the GBD and VCA domain.

Bacteria, plasmids, and cell lines

The EHEC dam mutant used in this study was derived from TUV93-0, a Shiga toxin-deficient version of the prototype O157:H7 strain EDL933 [44]. The EPEC bacterium was the prototype O127:H6 strain, JPN15/pMAR7. EPEC KC12, which contains the EPEC tir-cesT-eae operon replaced with the corresponding EHEC operon, has also been described [45]. A non-pathogenic strain of E. coli (MC1061) expressing EHEC intimin was also described elsewhere [31]. For yeast two-hybrid analyses, espF_U derivatives were generated by PCR from EDL933 genomic DNA and cloned into the EcoRI and BamHI sites of pGAD424 [28] to create fusions between an N-terminal GAL4AD and C-terminal 5myc tag. For transfections, espF_U fragments were subcloned into the KpnI and BamHI sites of pKC425 [46] to generate fusions between an N-terminal GFP-tag and C-terminal 5myc tag. For protein expression and purification, most espF_U derivatives were subcloned into the NdeI and XbaI sites of the vector pET16b (Novagen) to create fusions between an N-terminal His10-tag and C-terminal 5myc tag. Some EspF_U constructs contained only an N-terminal His-tag (Figure 8) [41]. For Tir-EspF_U hybrids, espF_U-myc derivatives were subcloned into the KpnI and XbaI sites of the HN-Tir-related vector pKC689 [31]. EspF_U truncations contained amino acids 1-88 (N), 80-384 (R1-6), 80-275 (R1-4), 80-228 (R1-3), 80-181 (R1-2), 80-134 (R1), 229-384 (R4-6), 276-384 (R5-6), 323-384 (R6), 80-314 (R′1-5), 221-314 (R′4-5), and 268-314 (R′5). For dominant negative transfections, rat N-WASP derivatives were generated by PCR with an N-terminal Flag-tag using domain boundaries described previously [28] and cloned into the KpnI-EcoRI sites of the vector pCDNA3 (Invitrogen). N-terminally GFP-tagged derivatives of the GBD were generated by subcloning into pKC425. Plasmids for expression of membrane-targeted HN-TirFL in mammalian cells and expression of the N-WASP GBD fused to the LexA DBD in pBTM116 have been described previously [28],[31]. Plasmids for expression of His-Flag-N-WASP and His-Myc-WIP in insect cells are described elsewhere (ADS and MDW, submitted). N-terminally His- or GST-tagged WASP GBD (residues 242–310), N-WASP_C (residues 193-501) and WASP GBD-VCA (residues 230–310 and 420–502 with a GGSGGS linker) constructs are described elsewhere [41]. For routine passage, all E. coli strains were grown in LB media at 37°C. Prior to infections, EHEC was cultured in DMEM+100 mM HEPES pH 7.4 in 5% CO₂ to enhance type III secretion. HeLa, Cos7, and murine fibroblast-like cells (FLCs) [47] were used interchangeably and cultured in DMEM+10% FBS at 37°C in 5% CO₂.

Transfections and infections

All transfections were performed as described previously [31]. Infections for 3 h with non-pathogenic E. coli expressing intimin [31] and EHEC or EPEC strains [44]–[45] have also been described. To cluster Tir, cells were treated with antibodies that recognize its extracellular domain prior to the addition of formalin-fixed S. aureus Pansorbin particles (Calbiochem) [25].

Immunofluorescence microscopy

Infected cells were fixed in 2.5% paraformaldehyde for 35 minutes and permeabilized with 0.1% Triton-X-100 in PBS as described previously [45]. Bacteria were identified with 1 µg/ml DAPI (Sigma), and F-actin was detected using 4 U/ml Alexa568-phalloidin (Molecular Probes). To visualize N-WASP derivatives, cells were treated with an anti-Flag M5 antibody (Sigma) and Alexa488 goat anti-mouse antibodies (Molecular Probes). To visualize HN-Tir derivatives, cells were treated with an HA.11 antibody (Covance) and Alexa488 or Alexa350 goat anti-mouse antibodies. To quantify the pedestal formation index in cells expressing high levels of N-WASP derivatives, which were identified by bright anti-Flag or GFP fluorescence, the percentage of cells harboring at least 10 adherent bacteria and 5 actin pedestals was measured. To quantify the pedestal index in cells infected with KC12, the percentage of cells harboring at least 10 adherent bacteria and 5 actin pedestals was measured. To quantify the pedestal index in cells treated with pansorbin particles, the percentage of HA-fluorescing cells harboring at least 10 adherent particles (the latter identified by virtue of their ability to bind fluorescently labeled secondary antibodies) and 5 actin pedestals was measured. At least 50 cells were examined per sample per experiment. Our previous work suggests that scoring of a pedestal index accurately reflects similar quantification methods that measure the fraction of bound bacteria that generate pedestals [25]. Cells expressing extremely high fluorescence levels of EspF_U were refractory to pedestal formation and were not included in these analyses. All scalebars are 1 µm in length.

EspF_U interaction assays

For yeast two-hybrid assays. EspF_U variants consisting of different combinations of the 6 repeats were fused to the Gal4 transcriptional activation domain, whereas the N-WASP GBD was fused to the LexA DNA-binding domain. Pairwise combinations of these fusion proteins were tested for interactions by measuring activation of a lacZ reporter, as described [28]. For some pulldown assays (Figure 4B), cobalt-chelate conjugated magnetic Talon particles (Invitrogen) were saturated with His-EspF_U-myc derivatives by incubating them with 75 µg/ml of each recombinant fragment for 1 h. After removal of unbound EspF_U, beads were incubated with brain extract for 1 h in 50 mM NaPO₄ pH 7.4, 150 mM NaCl, and 0.015% Triton X-100. Bound proteins were eluted by boiling in SDS-PAGE sample buffer. For other pulldown assays (Figure 4C), His-EspF_U-myc derivatives were incubated in brain extract at specific concentrations for 0.5 h and collected using an excess of Talon particles. Bound proteins were again eluted by boiling in SDS-PAGE sample buffer.

Immunoblotting

To prepare mammalian cell lysates, transfected cells were collected in PBS+2 mM EDTA and lysed in 50 mM Tris-HCl, pH 7.6, 50 mM NaCl, 1% Triton X-100, 1 mM Na₃VO₄, 1 mM PMSF, and 10 µg/ml each of aprotinin, leupeptin, pepstatin, and chymostatin (Sigma)), prior to mixing with SDS-PAGE sample buffer. Protein samples were boiled for 10 minutes, centrifuged, and analyzed by 10% SDS-PAGE prior to staining with Coomassie blue or transferring to nitrocellulose membranes and staining with Ponceau S. Membranes were blocked in PBS+5% milk (PBSM) before probing with N-WASP or GFP antibodies, as described previously [28]. Following washes, membranes were treated with secondary antibodies conjugated to alkaline phosphatase or horseradish peroxidase and developed using BCIP/NBT [45] or enhanced chemiluminescence [48].

EspF_U expression and purification

His-EspF_U-myc fusion proteins were expressed in E. coli BL21-Rosetta (Novagen) at 37°C in the presence of 0.1 mM IPTG for 3 h. Bacteria were lysed in 10 mM Tris pH 7.4, 150 mM NaCl, and protease inhibitor cocktail (Roche) using a cell disruptor (30kpsi, Constant Cell Systems). EspF_U was purified first using His-tag affinity for Ni-NTA-agarose beads (Qiagen) and eluted in lysis buffer containing 400 mM imidazole. To remove EspF_U degradation products, an anion exchange purification step, facilitated by the negatively charged 5myc-tag, was performed. EspF_U was bound to a HiTrap Q column (GE Healthcare) and eluted in 10 mM Tris pH 9.5 containing 1 M NaCl. Other EspF_U constructs (R′5, R′4-5, R1-5; Figure 8) with just an N-terminal His-tag were expressed in E. coli BL21-DE3 at 20°C in the presence of 1 mM IPTG for 16 h, and after Ni-NTA affinity purification the tag was cleaved with thrombin. Cleaved proteins were subjected to ion exchange and gel filtration chromatography to remove the tag and other impurities. Protein concentrations were estimated by Bradford assay (Bio-Rad).

Preparation of brain extracts

Brains obtained from freshly slaughtered pigs (Dalehead Foods Ltd, Linton, UK) were cleaned, sectioned, and resuspended in extraction buffer (0.1 M MES pH 6.8, 1 mM EGTA, 0.5 mM MgCl₂, 0.1 mM EDTA, 1 mM DTT, protease inhibitor cocktail), prior to homogenization using a Waring blender (2×15 s, 4°C). After clarification (7,000 g, 20 min, 4°C), extracts were filtered through cheesecloth and further clarified (11,000 g, 40 min, 4°C) prior to storage (−80°C). Extract to be subfractionated (60 ml) was dialysed against 2×5 L 20 mM Tris-Cl pH8, 2 mM MgCl₂, 5 mM EGTA, 1 mM EDTA, 0.5 mM DTT, 0.2 mM ATP, 2.5% glycerol at 4°C, prior to loading onto tandem HiTrap Heparin columns (2×5 ml), pre-equilibrated with dialysis buffer. Bound proteins were eluted using a 0–0.5 M KCl gradient in dialysis buffer (2 ml/min) and 20 3 ml fractions collected. Fractions 11–17 contained both Arp2/3 complex and N-WASP upon immunoblotting, and were combined, dialysed (as above), concentrated 5-fold (Centricon), and stored at −80°C.

N-WASP, WIP, and Arp2/3 expression and purification

His-tagged human N-WASP and WIP were expressed using recombinant baculovirus-infected High5 insect cells, as described in detail elsewhere (ADS and MDW, submitted). A recombinant N-WASP/WIP complex was purified by Nickel affinity chromatography followed by gel filtration chromatography to separate the complex from N-WASP or WIP alone. To maintain an autoinhibited N-WASP/WIP complex, freeze-thaw cycles and prolonged periods of storage on ice were kept to a minimum. Recombinant human Arp2/3 complex and native bovine Arp2/3 complex were purified as described previously [39],[41].

Pyrene-actin assembly assays

Assays utilizing brain extract contained samples supplemented with 2.5 µM skeletal muscle actin (10% pyrene-labeled), and polymerization was measured as described previously [49]. Assays using N-WASP/WIP complex contained 2.0 µM actin (7% pyrene-labeled) and 20 nM recombinant Arp2/3 complex, and polymerization was measured as described previously [48]. Assays using the N-WASP GBD-VCA contained 4.0 µM actin (5% pyrene-labeled) and 10 nM bovine Arp2/3 complex, and polymerization was measured as described elsewhere [41].

Isothermal titration calorimetry and gel filtration chromatography

Isothermal titration calorimetry was performed as described elsewhere [41]. Briefly, the WASP GBD was titrated into R′5, R′4-5, or R′1-5 in KMEI buffer (50 mM KCl, 1 mM MgCl₂, 1 mM EGTA, and 10 mM imidazole pH 7.0) plus 5 mM β-mercaptoethanol. Prior to gel filtration analyses, the N-termini of EspF_U fragments R′5 and R′4-5 were fluorescently labeled by dialysis against 100 mM sodium bicarbonate pH 8.3 and treatment with a five-fold molar excess of AlexaFluor647-carboxylic acid, succinimidyl ester (Invitrogen) at 4°C for 16 hours. Extra dye was removed by desalting chromatography and subsequent dialysis. Conjugation was confirmed by mass spectrometry. Labeling efficiency was estimated as >97% by the ratio of absorbance at 280 and 650 nm. Interactions between Arp2/3 complex and N-WASP_C in complex with EspF_U were examined using a Superdex 200 10/300 GL column (GE Healthcare) equilibrated in KMEI plus 1 mM dithiothreitol buffer.