Profilin is required for sustaining efficient intra- and intercellular spreading of Shigella flexneri.

The ability of Shigella to mediate actin-based motility within the host cell is a prominent pathogenic feature of bacillary dysentery. The ability is dependent on the interaction of VirG with neural Wiskott-Aldrich syndrome protein (N-WASP), which in turn mediates recruitment of Arp2/3 complex and several actin-related proteins. In the present study, we show that profilin I is essential to the rapid movement of Shigella in epithelial cells, for which the capacity of profilin to interact with G-actin and N-WASP is critical. In COS-7 cells overexpressing either mutated profilin H119E, which failed to bind G-actin, or H133S, which is unable to interact with poly-l-proline, Shigella motility was significantly inhibited. Similarly, depletion of profilin from Xenopus egg extracts resulted in a decrease in bacterial motility that was completely rescued by adding back profilin I but not H119E or H133S. In COS-7 cells overexpressing a N-WASP mutant lacking the proline-rich domain (Deltap) unable to interact with profilin, the actin tail formation of intracellular Shigella was inhibited. In N-WASP-depleted extracts, addition of Deltap but not full-length N-WASP was unable to restore the bacterial motility. Furthermore, in a plaque formation assay with Madin-Darby canine kidney cell monolayers infected by Shigella, Madin-Darby canine kidney cells stably expressing H119E, H133S, or Deltap reduced the bacterial cell-to-cell spreading. These results indicate that profilin I associated with N-WASP is an essential host factor for sustaining efficient intra- and intercellular spreading of Shigella.

The ability of Shigella to move within the host cell cytoplasm and into neighboring cells is an essential pathogenic feature of bacillary dysentery (1). Following internalization into epithelial cells, Shigella mediate actin polymerization at one pole of the bacterium by exploiting various host cell functions through which the bacteria gain a propulsive force and move in the host cell cytosol (1). Upon contact of the motile bacterium with the inner surface of the cell plasma membrane, a long membranous protrusion (filopodium) appears behind the bacterium, which is endocytosed by the neighboring cells, resulting in the bacterium being surrounded by a double membrane (2). Shigella then disrupts the membranes and escapes into the new host cytoplasm to multiply again. Thus, the ability of Shigella to elicit actin assembly in mammalian cells is crucial for spreading among colonic epithelial cells (3).
The ability of intracellular Shigella flexneri to evoke actinbased motility is mediated by the virG (icsA) gene product (4). VirG is an outer membrane protein, whose N-terminal half, designated as VirG-␣1, is exposed on the surface (5). The assembly of F-actin at one pole of the bacterium depends on the surface presentation of VirG-␣1 (6), including the asymmetric distribution of its protein (7). A similar actin-based motility has been reported in infection of epithelial cells by Listeria monocytogenes: the pathogen asymmetrically expresses ActA protein on the cell surface, where it mediates actin polymerization (8).
Actin polymerization mediated by VirG in mammalian cells requires various host proteins. Immunofluorescence of fixed infected cells has indicated an association of vinculin (2), plastin (9), filamin (9), ␣-actinin (10), vasodilator-stimulated phosphoprotein (VASP 1 ) (11), neural Wiskott-Aldrich syndrome protein (N-WASP) (12), Arp3, and zyxin (13) with the actin tail from motile S. flexneri. N-WASP and Arp2/3 complex have been indicated to play a key role in initiating actin polymerization (14,15). In the vicinity of the bacterial surface, a ternary complex composed of VirG, N-WASP, Arp2/3 complex is thought to be formed, where the Arp2/3 complex activated by N-WASP mediates actin nucleation with the aid of other host proteins (16). Based on the in vitro motility of N-WASP-coated, VirG-expressing Escherichia coli, Loisel et al. concluded that actin, Arp2/3 complex, actin depolymerizing factor/cofilin, and capping protein are sufficient to promote bacterial motility (17). In subsequent reconstitution experiments, they also indicated that profilin and ␣-actinin promoted the rate of movement of E. coli-expressing VirG (IcsA), but neither of the proteins are essential (17).
N-WASP is ubiquitously expressed in various tissues, including colon (18). N-WASP possess several distinctive domains: a pleckstrin homology domain that binds phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ), a GTPase-binding domain that binds Cdc42, a proline-rich (P) region that binds SH3 domains as well as profilin (19), a G-actin-binding verprolin homology (V) domain, an actin-depolymerizing protein cofilin homology (C) domain, and finally a C-terminal acidic (A) domain that binds Arp2/3 complex (20). N-WASP is a critical target for filopodium formation downstream of Cdc42 (21) and can be bound by profilin, required for rapid actin polymerization (19). Activation of N-WASP by Cdc42 unmasks the VCA region (22), which in turn leads to direct interaction with and activation of Arp2/3 complex, thus mediating actin nucleation (22).
Profilin, originally identified as a G-actin sequestering protein (23), has been indicated to be variously involved in modulating actin dynamism. In vitro, profilin can accelerate either actin assembly or the sequestering of actin depending on the presence of other actin binding proteins and on the state of the barbed ends of F-actin. Profilin accelerates the polymerization of actin from the G-actin pool at the free barbed ends of the actin filaments. Profilin also catalyzes the exchange of ADP to actin-bound ATP to promote actin assembly from the G-actin pool (24). On the other hand, when the barbed ends of the F-actin filaments are capped by proteins, such as capping protein, profilin acts as an actin sequestering factor that accelerate the depolymerization of F-actin or the inhibition of actin assembly.
Profilin can interact with various host components such as G-actin, PI(4,5)P 2 (25), poly-L-proline (26), and proteins with proline-rich sequences such as N-WASP (19), VASP (27), MENA (28), p140mDia (29), and Arp2/3 complex (30). Profilin exists in two isoforms in mammals, profilins I and II, and their distributions vary among tissues, including host species (31,32). Furthermore, the affinity of each isoform for the ligand proteins varies; in vitro, profilin I has a higher affinity for N-WASP (K d ϭ 60 nM) than does profilin II (K d ϭ 400 nM) (19). Although different functions for profilins I and II have been indicated, the biological significance of the profilin isoforms still remains obscure.
The VirG protein of S. flexneri interacts directly with N-WASP for actin-based motility, which leads to the formation of long filopodium (12). Suetsugu et al. (19) reported that the interaction of profilin I with the P-region of N-WASP was involved in rapid actin polymerization in filopodium formation, suggesting that binding of profilin to the P-region of N-WASP plays a functional role in filopodium extension and probably the elongation of actin tail generated from motile Shigella. However, H133S-mutated profilin that was unable to bind to poly-L-proline was shown to enhance the rate of movement of E. coli-expressing VirG in profilin-depleted cell extracts to the exact same extent as bovine profilin, suggesting that the interaction of profilin with poly-L-proline sequences, including N-WASP, is not involved in the enhancement of actin-based bacterial motility (16).
In this context, we wished to clarify the role of profilin I in the VirG⅐N-WASP-mediated Shigella motility in mammalian cells. The present study provides evidence that profilin I is an important host component for sustaining high speed Shigella movements within the primary infected host cells and into the adjacent cells and that the functional interactions of profilin I with N-WASP and G-actin are both involved in the actin-based bacterial motility in mammalian cells.
Sf9 insect cells used for baculovirus expression were cultured in Sf-900 II SFM medium (Life Technologies, Inc.) containing 10% FCS at 27°C with vigorous shaking.
Antibodies-The anti-N-WASP-specific rabbit polyclonal antibody was described previously (34). Anti-profilin I antibody was produced by immunizing rabbits with GST-profilin I. For purification of anti-profilin I antibody, the antiserum was incubated with GST-profilin II-conjugated glutathione-Sepharose 4B beads to remove the Ig fraction that reacted with GST-profilin II, then the specific antibody for profilin I was purified by affinity column chromatography. The anti-VirG-specific rabbit polyclonal antibody was described previously (35). The anti-Myc antibody was from Santa Cruz Biotechnology. The anti-actin mouse monoclonal antibody and anti-ZO-1 rat monoclonal antibody were from Chemicon International Inc. The anti-E-cadherin mouse monoclonal antibody was from Transduction Laboratory. The secondary antibodies linked to fluorescein, peroxidase-conjugated anti-mouse IgG antibody, and peroxidase-conjugated protein A were from Sigma. The secondary antibody linked to Cy5 was from Amersham Pharmacia Biotech. Rhodamine-labeled phalloidin was from Molecular Probes Inc.
The histidine 133 to serine-substituted (H133S) profilin was generated from pGEX-2T-profilin I using a Unique Site Elimination mutagenesis kit (Amersham Pharmacia Biotech). Expression and purification of GST-profilins (wild-type, histidine 119 to glutamic acidsubstituted (H119E) mutant and H133S mutant profilin I) were carried out as described previously (19). Profilins without GST were obtained from GST-profilins by thrombin digestion as described previously (36).
Recombinant baculovirus expressing rat N-WASP was prepared by using the BAC-TO-BAC expression system (Life Technologies, Inc.) (20). The construction of ⌬p mutant N-WASP (lacking amino acids 276 -401) was carried out as follows; the cDNA coding for the Nterminal (nucleotides 1-825) region of ⌬p mutant N-WASP cDNA was amplified by polymerase chain reaction using a plasmid encoding fulllength N-WASP on pBluescript (Stratagene) as a template. The C terminus region (nucleotides 1206 -1518) was obtained from full-length N-WASP on pBluescript by digestion with PstI and EcoRI to delete the DNA fragment coding nucleotides 1-1205. These fragments were ligated through PstI sites in the polymerase chain reaction primer. The resulting cDNAs were ligated to pFASTBAC1 plasmid (Life Technologies, Inc.) for baculovirus expression. Purification of N-WASP proteins from cell lysates of infected Sf9 cells was performed as described previously (12).
Transient Expression of Profilin, N-WASP, and Mutants in COS-7 Cells-The plasmid containing pEF-BOS-myc-profilin I was constructed as described (19). The cDNAs for H119E mutant, H133S mutant, or wild-type profilin I were cloned into pEGFP-C1 plasmid (CLONTECH) for expression of green fluorescence protein (GFP)tagged profilins in cultivated cells. The constructs of full-length or ⌬p mutant of N-WASP on pcDL-SR␣ were described previously (19). Recombinant plasmids for transfection were prepared from E. coli using a plasmid Mini Kit (Qiagen). A total of 7 ϫ 10 5 COS-7 cells was mixed with each purified plasmid (20 g) and transfected by electroporation. The cells were reseeded and cultured for 48 h before bacterial infection.
Infection of Cultured Cells and Immunofluorescence Microscopy-Cells were seeded on coverslips and infected with S. flexneri or L. monocytogenes as described previously (12). After fixation with 4% paraformaldehyde in phosphate-buffered saline, the coverslips were permeabilized and blocked. Immunofluorescence staining was performed by incubation with anti-N-WASP or anti-Myc antibodies. FITCconjugated secondary antibody was used to visualize Myc, CyDye-conjugated secondary antibody was used to visualize N-WASP, and rhodamine-phalloidin was used to visualize F-actin. To avoid the bleedthrough of the signals, each fluorescence image was collected sequentially by a single channel excitation using a MicroRadiance confocal scanning system equipped with image processing software (LaserSharp Radianceplus version 3.2, Bio-Rad). Accumulation of F-actin around the intracellular bacteria was defined as areas where the intensity was higher than an arbitrary threshold of 80 estimated in non-infected cells using LaserSharp Radianceplus.
Motility Rate Assay of S. flexneri in COS-7 Cells-COS-7 transfectants were reseeded in 35-mm glass-base dishes (Mat Tek Corp.) and cultured for 48 h before bacterial infection. Infection with S. flexneri YSH6000 was carried out as described above. At 1-2 h after infection, movement of S. flexneri was observed with an Axiovert 135 microscope (Zeiss) equipped with a SenSys 1400 CCD camera (Roper Scientific) in a chamber maintained at 37°C with a 5% CO 2 atmosphere. The GFPoverexpressing cells were observed in fluorescence, and the bacteria were observed in phase-contrast mode. The bacterial movements in GFP-overexpressing cells were recorded as time-lapse images, and velocity was analyzed using IPLab Spectrum software (Signal Analytics Corp.). The non-motile bacteria were omitted from the records. Differences in migration velocities were analyzed using the unpaired Student's t test.
In Vitro Binding Assay Using GST Fusion Protein-Recombinant GST-human profilin I or GST alone immobilized on glutathione-Sepharose beads was incubated with 0.4 M full-length or ⌬p mutant of recombinant N-WASP in Tris-buffered saline (TBS) containing 0.5% Triton X-100 (TBS-TX) at 4°C for 1 h. Samples were washed three times in TBS-TX and subsequently incubated with 0.4 M VirG (␣1) at 4°C for 1 h. After another wash, samples were subjected to Western blot analysis using anti-VirG or anti-N-WASP antibodies. COS-7 transfectants of full-length or ⌬p mutant (⌬p) of N-WASP on pcDL-SR␣ plasmid or pcDL-SR␣ plasmid alone (vector) were grown in 100-mm dishes and lysed in ice-cold radioimmune precipitation buffer as described previously (12). For GST-␣1 pull-down assay, the cell lysates were incubated with recombinant human profilin I (1 mM) and GST-␣1 beads overnight at 4°C. Samples were washed four times in radioimmune precipitation buffer and subjected to Western blot analysis with anti-N-WASP and anti-profilin I antibodies.
Actin Tail Assay in Xenopus Egg Extracts-Meiotically arrested cytoplasmic extracts of Xenopus laevis eggs were prepared as described previously (37). G-actin was purified from rabbit skeletal muscle as described (38). Purified G-actin was covalently labeled with tetramethylrhodamine iodoacetamide (Molecular Probes) as an actin tail tracer. Depletion of N-WASP from Xenopus egg extracts using anti-N-WASP antibody was carried out as described previously (12). Poly-L-proline peptides (Sigma) were conjugated with CNBr-activated Sepharose FF (Amersham Pharmacia Biotech) as described previously (39). For control beads, the N-terminal-activated residues of CNBr-activated Sepharose FF beads were blocked by incubation with blocking buffer (0.1 M glycine, 50 mM Tris-HCl, pH 8.0). A 20-l volume of beads was washed twice with 1 ml of XB (100 mM KCl, 50 mM sucrose, 5 mM EGTA, 2 mM MgCl 2 , 0.1 mM CaCl 2 , 10 mM HEPES, pH 7.7) and incubated with 30 l of extract for 1 h at 4°C. The supernatant was removed by centrifugation and treated as the poly-L-proline-depleted extract. Aliquots of the pellets and supernatant were analyzed by Western blotting. Motility assay was carried out as described (40) using a microscope equipped with a CCD camera. The bacterial movement and fluorescence intensity during a 10-to 60-min period were recorded as time-lapse images and analyzed using IPLab Spectrum software (Signal Analytics Corp.). Total fluorescence intensity was determined by multiplying the average pixel intensity by the pixel area. The fluorescence intensity value of the actin tail was calculated by subtraction of the intensity of a background area from that of the selected area. The illumination level of the observation was in the range of 10 -2000 counts/pixel in which the CCD camera responds linearly.
Preparation of MDCK Cell Lines Stably Expressing Profilin, N-WASP, and Mutants-Transfection of pEGFP-C1 plasmid containing profilin I (or its mutants) or pcDL-SR␣ plasmid containing N-WASP (or its mutant was carried out by electroporation, and cell clones were isolated based on resistance to geneticin. The expression levels of profilin and N-WASP in stable transformants were examined by Western blot analysis using the whole cell lysates. For localization of actin filaments, GFP-tagged proteins, N-WASP, ZO-1, and E-cadherin, immunofluorescence microscopy was performed as described above. Plaque Forming Assay-MDCK cells were seeded in 24-well tissue culture plates and incubated for 2 days at 37°C in a 5% CO 2 atmosphere. Each well was infected with S. flexneri YSH6000 at a multiplic-ity of infection of 10. After 4 h, cells were washed with Hanks' balanced salt solution and Dulbecco's modified Eagle's medium supplemented with 10% FCS, 100 g/ml gentamicin, and 60 g/ml kanamycin. At 3 days post-infection, the average size of plaques on cells was measured by a phase-contrast microscope equipped with a CCD camera using the IPLab Spectrum software. , and pEGFP-C1-profilin I H133S mutant (D) were infected with S. flexneri YSH6000. The motility rate of intracellular Shigella in GFP-expressing cells at 1 through 2 h after infection was monitored by fluorescence and a phase-contrast microscope equipped with a CCD camera. The distribution of the motility rates was plotted. In each case, ϳ55 intracellular bacteria were measured.

Recruitment of Profilin by Intracellular
Shigella-To investigate the association of profilin with the actin tail of intracellular S. flexneri, we constructed COS-7 transfectants overexpressing Myc-tagged human profilin I. A cDNA encoding Myctagged human profilin I was cloned into pEF-BOS vector, and the resulting plasmid (pEF-BOS-myc-profilin I) was transfected into COS-7 cells. The Myc-tagged protein localized to sites of actin-cytoskeletal reorganization (data not shown). The transfectants were then infected with a wild-type S. flexneri strain (YSH6000), and the localization of Myc-profilin I in the COS-7 transfectants was examined at 2 h post-infection by double immunostaining. As shown in Fig. 1, Myc-profilin I was incorporated into the front of and throughout the actin tail generated by intracellular bacterium. Similar results were obtained by single staining of Myc-profilin I or double immunostaining of Myc-profilin I and F-actin in HeLa and Caco-2 cells (data not shown).

Effect of Overexpression of Mutant Profilin I on Shigella
Motility-To investigate whether the association of profilin with intracellular Shigella plays a functional role in bacterial motility, COS-7 cells overexpressing GFP-profilin I H119E or H133S mutants were infected by S. flexneri YSH6000 and examined for the effect on bacterial motility. The H119E mutant, possessing a histidine 119 to glutamate substitution, was unable to bind to G-actin but not to other ligands such as p140mDia, gephyrin, MENA, N-WASP, and VASP (19), whereas the H133S mutant, containing a histidine 133 to serine substitution, was unable to interact with proteins possessing proline-rich sequences (41), including N-WASP (data not shown). Immunoblotting with anti-profilin I antibody confirmed that each mutant GFP-profilin I was expressed at a much higher level in the transfectants than the native endogenous profilin I (data not shown). The distribution of GFPprofilin in the COS-7 transfectants was similar to that of the Myc-tagged profilin in COS-7 cells (data not shown). The transfectants were then infected with S. flexneri YSH6000, and the motility rate was assayed at 1-2 h after infection using fluorescence microscopy. As shown in Fig. 2 To confirm that the decreased bacterial motility was not due to the fused GFP moiety to profilin or high concentrations of GFP-profilin I in COS-7 cells, we measured the bacterial motility in COS-7 cells overexpressing GFP-profilin I. Overexpression of GFP-profilin I slightly increased the motility of bacteria to 17.72 m/min (S.E. ϭ 1.54, n ϭ 59, p Ͻ 0.05), suggesting that profilin is functionally involved in accelerating the motility of intracellular Shigella.
Depletion of Profilin from Xenopus Egg Extracts Affects VirGdirected Actin Assembly and Bacterial Motility-To further clarify the role of profilin in the bacterial motility in mammalian cells, we depleted profilin from Xenopus egg extracts using poly-L-proline beads and tested for the capacity to support actin tail formation of VirG-expressing E. coli (virG gene-encoding pD10-1-carrying MC1061 OmpT::Km). Poly-L-proline-Sepharose beads or glycine (N-terminal blocked)-Sepharose beads (control beads) were incubated with the extracts at 4°C for 1 h, and the amounts of profilin in the depleted extracts were examined by immunoblotting. Poly-L-proline beads removed all detectable profilin from extracts and some amounts of actin (Fig. 3A). The profilin-depleted extracts had decreased actin assembly compared with the extracts treated with control beads (Fig. 3C, b and c), and bacterial motility was reduced by approximately 60% in depleted extracts (Fig. 3B). When recombinant human profilin I plus G-actin was added to approximately physiological concentrations (ϳ1.0 and ϳ3.3 M, respec- tively), bacterial motility and the formation of actin tails were restored ( Fig. 3B and C, f), although recombinant human profilin I or G-actin alone could not rescue the activities at all ( Fig.  3B and C, d and e). Under the same conditions, however, activity was not rescued by addition of recombinant mutant human profilin I H119E or H133S ( Fig. 3B and C, g and h). These results further indicate that the abilities of profilin to interact with actin and proteins possessing proline-rich sequence are functionally involved in the acceleration of motility of intracellular Shigella.
Association of Profilin, N-WASP, and VirG-To elucidate whether the observed profilin functions in Shigella motility are mediated via the interaction with N-WASP, we examined the association of profilin I, N-WASP, and VirG in vitro. A GSThuman recombinant profilin I immobilized on glutathione-Sepharose beads (or GST alone immobilized on glutathione-Sepharose beads) and recombinant N-WASP (or ⌬p mutant N-WASP) were incubated first at 4°C for 1 h and then in the presence of a recombinant VirG protein at 4°C for 1 h. The ⌬p mutant of N-WASP has a deletion at amino acids 276 -401 that encompasses the proline-rich domain, which is essential for binding to profilin (19). We first checked whether the ⌬p mutant of N-WASP enhanced the actin nucleation activity of Arp2/3 complex in a Cdc42-activated fashion identical to the full-length N-WASP. In brief, using an in vitro pyrene actin polymerization assay (42), recombinant ⌬p N-WASP could stimulate actin polymerization in the presence of purified bovine Arp2/3 complex at the same rate as the full-length N-WASP, and Cdc42 could activate ⌬p N-WASP⅐Arp2/3 complex as well as full-length N-WASP⅐Arp2/3 complex (data not shown). The proteins precipitated by the beads were examined by immunoblotting with anti-N-WASP antibody or anti-VirG antibody. Recombinant VirG only precipitated with the profilin I beads in the presence of full-length N-WASP and not in the presence of the ⌬p mutant (Fig. 4A). Because the ⌬p mutant could not bind profilin, while the GST-human profilin I could not bind the recombinant VirG (Fig. 4A), the precipitation of VirG by the human profilin I beads was the result of its interaction with N-WASP. A GST pull-down assay was performed in COS-7 cells transfected with pcDL-SR␣⅐N-WASP (full-length) or pcDL-SR␣⅐⌬p N-WASP (⌬p mutant) plasmid (19), and the cell lysates were incubated with recombinant human profilin I and GST-VirG beads or GST beads. Immunoblotting with anti-N-WASP antibody or anti-profilin I antibody revealed that, although profilin I was precipitated in the presence of fulllength N-WASP and GST-VirG beads, it did not bind in the presence of ⌬p mutant (Fig. 4B), confirming that profilin can associate with VirG through binding to N-WASP.
Overexpression of ⌬p N-WASP Mutant in COS-7 Inhibits Assembly of Actin Tail from Intracellular Shigella-To investigate the ability of N-WASP to recruit profilin, COS-7 cells overexpressing full-length N-WASP or ⌬p mutant together with Myc-profilin I were infected with S. flexneri YSH6000 and examined by triple immunostaining with Cy5-labeled rabbit anti-N-WASP, FITC-labeled anti-Myc antibodies, and rhodamine-phalloidin. After 120 min of infection, N-WASP was highly concentrated together with profilin I at one pole of the bacteria and was also seen at the actin tail (Fig. 5A, a-c). In contrast, although ⌬p mutant N-WASP accumulated at the site of VirG deposition on bacteria in COS-7 cells overexpressing ⌬p mutant and Myc-profilin I (Fig. 5A, d), Myc-profilin I was barely detected on the surface of bacteria (Fig. 5A, e). The effect of overexpression of the ⌬p mutant on assembly of the actin tail by S. flexneri in COS-7 cells was further examined by counting the number of actin assemblies associated with the intracellular bacteria. As shown in Fig. 5C, after 120 min of infection, approximately 94% (93.5 Ϯ 3.3%, n ϭ 291) of intracellular bacteria possessed an actin clot or tail in cells overexpressing full-length N-WASP, compared with 12% (11.8 Ϯ 8.0%, n ϭ 367) in cells overexpressing ⌬p mutant N-WASP. The overexpression of ⌬p or the full-length N-WASP had no effect on the actin association in intracellular L. monocytogenes (Fig. 5C). Therefore, the overexpression of ⌬p N-WASP mutant greatly inhibited the formation of an actin tail by intracellular S. flexneri.
Significance of N-WASP in Recruitment of Profilin in Shi- gella Motility-N-WASP was immunodepleted from Xenopus egg extracts with anti-N-WASP antibody immobilized on protein A beads at 4°C for 1 h. Upon depletion of N-WASP, the generation of an actin tail from E. coli MC1061 ompT::Km carrying pD10-1 was abrogated, whereas when full-length N-WASP was added at approximately physiological concentrations (ϳ45 nM) into the depleted extract, most of the bacteria formed an actin tail (Fig. 6C, a and b). When ⌬p mutant (ϳ45 or 450 nM) was added to the depleted extract, none of the bacteria formed actin tails, and only had a weak actin clot around the bacterial body ( Fig. 6A and C, c and d). Although the bacterial motility in the depleted extracts was greatly restored by full-length N-WASP, it was not restored by addition of ⌬p mutant (Fig. 6A). These results thus strongly indicate that the association of profilin with N-WASP is involved in promoting elongation of the actin tail from motile Shigella.
Effect of a Dominant Interfering Profilin Mutant or ⌬p N-WASP on Cell-to-Cell Spreading of Shigella-To reveal the role of profilin in the intercellular spreading of Shigella, we constructed MDCK cell lines stably expressing wild-type profilin I, H119E mutant, or H133S mutant and investigated the ability of the bacteria to spread based on the size of plaques, a consequence of the actin-based motility, including the continuous intercellular spreading of Shigella. As represented in Fig. 7, A-D, and Table I, the average size of plaques formed by S. flexneri YSH6000 in MDCK monolayers expressing H119E or H133S mutants at 3 days post-infection was significantly smaller than that of the mock control or the wild-type profilin I, further confirming the involvement of profilin I in the bacterial spreading. We subsequently constructed MDCK cell lines stably expressing full-length N-WASP or ⌬p N-WASP mutant and examined the MDCK cell monolayers infected by YSH6000 for the size of plaques. The average size of the plaques in MDCK monolayers expressing ⌬p mutant at 3 days post-infection was less than 13% of that for the mock control or fulllength N-WASP (Fig. 7, E-G, and Table I). Because we found no alteration in cell-to-cell junctions as judged by immunostaining with anti-E-cadherin or anti-ZO-1 antibody (data not shown), these results strongly indicate that profilin plays an important role in promoting the efficiency of Shigella to move from one cell to another.

DISCUSSION
In this study, we have investigated whether or not profilin I was functionally involved in the movement of S. flexneri in mammalian cells and concluded that profilin I is an essential host factor for gaining high speed bacterial movement, for which the ability of profilin I to interact with actin as well as proteins containing proline-rich sequences such as N-WASP is critical. Our conclusion is based on the following results; (i) profilin I was colocalized with the actin tail generated from rapidly moving Shigella in COS-7 cells, (ii) COS-7 cells overexpressing either of the profilin I mutants, H119E (defective in binding actin) or H133S (defective in binding proline-rich sequences) interfered with bacterial motility, (iii) depletion of profilin from Xenopus egg extracts decreased bacterial motility, which was rescued by adding back wild-type profilin I but not H119E or H133S mutant profilin I, (iv) overexpression of dominant-negative N-WASP mutant (⌬p N-WASP) defective in profilin I interaction in COS-7 cells resulted in a decrease in actin tail formation from intracellular Shigella, and (v) immunodepletion of N-WASP from Xenopus egg extracts caused an arrest of bacterial motility, which was rescued by exogenously adding N-WASP but not ⌬p N-WASP.
In mammalian cells, two isoforms of profilin, profilin I and profilin II, exist; profilin I is more ubiquitously expressed and abundant than profilin II, whereas profilin II is predominantly expressed in neuronal cells (31,32). In addition, profilin I is a better ligand than profilin II for N-WASP in vitro (19), and N-WASP plays a crucial role in the actin-based motility of Shigella (12). Thus, we focused in this study on the role of profilin I. Initially, although we attempted to investigate the localization of profilin I at one pole of intracellular S. flexneri or in the long actin-tail generated in rapidly moving bacterium using immunostaining with anti-profilin I antibody, we could not detect any specific signals of endogenous profilin I because of the low affinity of the antibody used and the high background of profilin I (data not shown). Therefore, we exploited Myc-tagged profilin I and carried out immunostaining of intracellular Shigella in COS-7 cells overexpressing Myc-tagged profilin I. The results showed that the Myc-tagged profilin I colocalized in the actin tail generated by motile S. flexneri at all stages. The association of profilin I would not be a consequence of nonspecific binding, but rather, functional involvement in Shigella motility. Overexpression of GFP-H119E mutant profilin I, a dominant negative profilin I mutant defective in actin binding but retaining the ability to interact with other known ligands such as p140mDia, gephyrin, MENA, VASP, and N-WASP (19), greatly inhibited bacterial motility, suggesting that G-actin binding to endogenous profilin I was functionally involved in the bacterial motility (Fig. 2). The inhibitory effect by GFP-H119E was not ascribed to the fusion of the GFP moiety with the N-terminal profilin I, because GFP-profilin I (wild-type) colocalized in the actin tail and had almost the same actin and poly-L-proline binding ability as did profilin I as examined by in vitro binding assay (data not shown). Indeed, on overexpression of GFP in COS-7 cells, intracellular S. flexneri moved as rapidly as on overexpression of wild-type profilin I (Fig. 2).
To clarify whether the ability of profilin I to interact with N-WASP was also involved in Shigella motility, we constructed H133S-mutated profilin I, because H133S was known to be deficient in binding poly-L-proline (41). The H133S and GFP-H133S constructions were confirmed to be deficient in binding poly-L-proline and N-WASP. 2 In COS-7 cells overexpressing H133S, the motility of intracellular S. flexneri was greatly decreased (Fig. 2), suggesting that the ability of profilin to interact with proline-rich sequences such as N-WASP was involved in supporting high speed bacterial motility. This notion was supported by the motility assay of VirG-expressed E. coli using depletion/adding back of profilin I in Xenopus egg extracts. In our experiments, adding back pure recombinant wildtype profilin I alone did not restore the decrease motility at all 2 H. Mimuro, unpublished data.  (Fig. 3), however, addition of purified G-actin plus wild-type profilin I did recover the original motility rate of VirG-expressed E. coli. The requirement for supplement of G-actin in profilin depletion/add-back experiments was consistent with a previous report on Listeria motility in Xenopus egg extracts, in which addition of profilin plus G-actin but not profilin alone allowed Listeria to move rapidly in the extract (37). Thus, the pool of G-actin available for elongating actin filaments from motile Shigella would largely be associated with profilin in Xenopus egg cells as has been indicated for Acanthamoeba (43). In this regard, it is worth pointing that Egile et al. (16) have investigated the role of profilin in VirG⅐N-WASP⅐Arp2/3-mediated bacterial motility using platelet extracts. In the experiment, they exploited N-WASP-coated E. coli carrying pHS3199 (pUC8 possessing a cloned virG gene) and tested for the effect of profilin depletion on the bacterial motility. The results showed that, although the bacterial motility rate in the original extract was 2.5 m/min, in the profilin-depleted extract the motility was 1.3 m/min. When they added back wild-type profilin alone or H133S mutant profilin alone at approximately physiological concentrations into the depleted extract, the motility rate was 1.7 m/min. Based on the results, they concluded that profilin partly enhances actin-based motility, and that the ability of profilin to interact with proline-rich protein is not involved in the bacterial movement (16). Although the reason for the discrepancy between Egile et al. and the present study is unclear, we assume that the partial restoration of bacterial motility by wild-type profilin alone is due to a lack of supplement of G-actin.
Although the manner in which profilin interacts with the proline-rich sequence in the regulation of actin dynamism in living cells is still poorly understood (44), study of the actinbased motility of L. monocytogenes has provided some insight into the positive role of profilin (37). Profilin not only contributes to the efficiency of Listeria motility in cell extracts by increasing the bacterial speed but also works in cooperation with the Ena/VASP proteins to significantly support the actinbased motility (45). Geese et al. (46) recently reported that profilin II is associated with motile Listeria but not with a slow moving Listeria mutant that is unable to recruit Ena/VASP proteins. Indeed, earlier work (47) had indicated that the speed of Listeria was proportional to the rate of actin polymerization and that profilin stimulated the growth of actin filaments. Although Ena/VASP has no functional role in Shigella motility in mammalian cells (16), N-WASP is critical in mediating the actin-based motility of Shigella (12).
Recent studies have indicated that Shigella motility is mediated by a two-step process: nucleation of the actin filament and subsequent elongation of the actin tail. In the actin nucleation step, N-WASP activated by Cdc42 and partly VirG itself seems to be able to recruit and activate Arp2/3 complex, thus mediating actin nucleation (16,42). Once the VirG⅐N-WASP⅐Arp2/3 complex in the vicinity of the bacterial surface is formed, the elongation of actin filaments, including their stabilization seems to constantly be mediated by the activated Arp2/3 complex with the aid of other host components such as actin depolymerizing factor/cofilin, capping proteins, ␣-actinin, and profilin, thus gaining propulsive force for supporting rapid movements of intracellular Shigella (17). Although the exact role of profilin in the elongation of actin filaments is still to be elucidated, the ability of profilin I to interact with N-WASP through its P-region could be critical. In fact, we showed the direct binding of profilin with the P-region of N-WASP associated with VirG in vitro (Fig. 4). The significance was confirmed in two different in vivo assay systems. In COS-7 cells overexpressing full-length or ⌬p mutant N-WASP together with Mycprofilin I, although Myc-profilin I was colocalized with N-WASP by associating with the actin tail generated by motile Shigella, the association of Myc-profilin I was not observed when overexpressed ⌬p mutant N-WASP or even ⌬p mutant was colocalized with VirG at one pole of the bacterium (Fig.  5A). Consistent with this, overexpression of ⌬p mutant in COS-7 cells greatly decreased the bacterial actin tail formation compared with that in COS-7 cells overexpressing full-length N-WASP (Fig. 5C). A similar inhibitory effect on VirG-expressed bacterial motility by ⌬p mutant was seen in N-WASPdepleted Xenopus egg extracts (Fig. 6), in which the addition of full-length N-WASP to the depleted extract recovered bacterial motility, whereas the addition of ⌬p mutant did not. The proline-rich region of N-WASP binds not only profilin but also the  adapter protein Ash/Grb2 through its SH3 domain (19). However, Ash/Grb2 was not recruited at all at the actin tail of motile Shigella-infecting mammalian cells (data not shown). Although host factors other than profilin I may participate in the binding to the P-region of N-WASP and affect the bacterial motility, we presume that the high affinity binding of profilin I to N-WASP plays an important role in supporting the rapid movements of intracellular Shigella.
Profilin is incorporated not only into the front but also throughout the actin tail generated by moving Shigella in infected host cells (Fig. 1). Because profilin I interacts with the Arp2 subunit of the Arp2/3 complex, albeit with less affinity than N-WASP (48), and Arp2/3 localizes in the actin tail (16), the localization of profilin along the actin tail would be reflected by interactions with the Arp2/3 complex. The profilin associated with Arp2/3 may be involved in accelerating polymerization and blanching of actin filaments in vivo (49). Profilin or profilin-actin interacting with N-WASP at the surface of motile Shigella may facilitate conveyance of profilin to the Arp2/3 complex at the Y-junction of the dendritic actin filament network in which rapid elongation of actin filaments would be promoted by profilin.
It is proposed that 60 -180 M actin-ATP would be needed to sustain a filament growth rate of 0.5-1 m/s for the actin-based motility of Shigella as well as the leading edge of the cell (50). Because a high local concentration of actin-ATP would be needed within the actin-filament polymerization area, it seems reasonable that profilin and/or profilin-actin would be recruited by N-WASP at the barbed end of actin filaments, where rapid elongation would be facilitated. Then, G-actin would be shuttled to the growing barbed end, which would be recruited such as via VirG-associated vinculin (6,51,52) or N-WASP (16) in close vicinity to the bacterium surface.
The essential role of profilin in supporting rapid intracellular as well as intercellular movement of Shigella was conclusively demonstrated in the cell-to-cell spreading assay using MDCK cells stably expressing H119E, H133S, or ⌬p N-WASP, where Shigella motility, including intercellular movement, was greatly inhibited as judged by the size of plaques formed in MDCK monolayers ( Fig. 7 and Table I). The result is especially important, because the size of plaques has been shown to be reflected by changes in the bacterial motility rate (42). Indeed, expression of either of the dominant negative profilin mutants or the dominant negative N-WASP mutant had a serious effect on the bacterial spreading (Fig. 7), a prominent pathogenic feature of Shigella.