Cell culture.

Human colonic epithelial T84 cells (ATCC, Manassas, VA) were grown and maintained in culture in Dulbecco's modified Eagle's medium (DMEM)/Ham's F-12 medium (1:1) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin as we previously described (32, 33). All media were obtained from Invitrogen. Cell monolayers (passages 27–45) were grown on polycarbonate inserts with 0.4-μm pore size (Costar, Cambridge, MA) or in plastic six-well plates for 7–14 days. Experiments were performed on confluent monolayers with transepithelial electrical resistances > 1,500 Ω·cm².

Human tissue specimens.

Samples of archived frozen human tissue included normal distal and proximal colon (n = 6). The samples were coded so no patient identifiers were linked to the specimens. This study was exempted under the Code of Federal Regulations Title 45 Section 46.101(b) by the Hopkins Institution Review Board.

Infection of T84 cells by EHEC strain and treatments with pharmacological agents.

Following a published protocol (33), we inoculated T84 cells apically with different concentrations of EHEC strain EDL933 modified to be Stx-negative and incubated them at 37°C in 5% CO₂ for up to 4 h. Fluorescently labeled Stx1-AlexaFluor680 (0.2 μg/ml) was added apically at the time of infection as were inhibitors, such as pirl-1 (0.5 μM), blebbistatin (50 μM), calyculin A (5 nM), Cdc42 inhibitory peptide (40 μg/ml), HOE694 (50 μM), and S3226 (10 μM). After 4 h, the cells were washed three times with cold PBS and fixed for immunofluorescence or lysed in RIPA buffer [1% Triton X100, 0.5% deoxycholic acid, 0.1% SDS, 50 mM Tris·HCl, pH 7.4, 150 mM NaCl containing 0.5 mM Na₃VO₄ and protease inhibitor cocktail 1:1,000 (Sigma P8340)] and centrifuged at 20,000 g at 4°C for 15 min for immunoblotting.

Lentiviral transduction of T84 cell with Cdc42 shRNA constructs.

Four sequence-verified short hairpin RNA (shRNA) lentiviral plasmids in a hairpin-pLKO.1 puromycin vector for Cdc42 gene silencing in human cells were obtained from The RNAi Consortium (TRC) library (http://www.broad.mit.edu/genome_bio/trc/rnai.html) through the Johns Hopkins HiT center from Open Biosystems (Huntsville, AL) and were used to generate lentiviral transduction particles as we previously described (33).

Detection of intracellular Stx1 in vitro.

The relative fluorescence intensity of Stx1–680 in 150 μg protein in total cell lysates, which corresponds to the intracellular Stx1, was measured in triplicate as we described (33). Alternatively, lysates were subjected to SDS-PAGE and the polypeptides transferred to nitrocellulose membranes. The relative fluorescence intensity of the Stx1–680 band was measured by Odyssey infrared imaging scanner (33) and normalized to the fluorescence intensity of GAPDH obtained by immunoblotting.

Detection of Stx1 transcytosis in vitro.

Cells grown on polycarbonate inserts were incubated with 0.2 μg/ml Stx1–680 in the absence (basal transcytosis) or presence of EDL933 bacteria (stimulated transcytosis) for the times indicated in the figure legends. At the end of the incubation, inserts were removed, 100-μl samples of media from the lower chamber containing transcytosed toxin were collected, and the relative fluorescence intensity of Stx1 was measured in triplicate as we previously described (33). Stx1 fluorescence intensity in conditioned media was normalized to fluorescence intensity of conditioned media from cells not infected with EHEC and not exposed to Stx1.

For SDS-PAGE experiments the basal-conditioned media containing transcytosed Stx1-Alexa680 from control and experimental monolayers were concentrated using Amicon Ultra Centrifugal Filter Devices (Millipore) to a final volume of ~150 μl as we have described (29). The supernatant samples (40 μl) were subjected to SDS-PAGE, transferred to nitrocellulose, and analyzed for the presence of fluorescent Stx1 by infrared imaging scanner.

Rabbit model of EHEC-induced colitis.

A rabbit cecal model of EHEC-induced colitis was used as described (33, 47). Briefly, the enteroadherrent rabbit E. coli pathogen RDEC-1, which forms attaching/effacing (A/E) lesions similar to those in EHEC-infected humans, was transduced with Stx1-converting phage ΦH19A to express Stx1 and given by gavage. To discriminate between Stx1-specific intestinal epithelial changes and the changes related to enteric colonization, we infected male New Zealand White rabbits (2.5–5 kg, Myrtle's Rabbitry, Thompsons Station, TN) with 10¹⁰ RDEC-1 (n = 2), 10¹⁰ Stx1-producing RDEC-H19A (n = 3) or PBS (n = 2). Experiments with animals were performed at the University of New Mexico, School of Medicine using a protocol approved by the University of New Mexico Animal Use Committee. Three days after challenge, animals were euthanized, and cecal tissues from control and experimental rabbits were rinsed with ice-cold PBS, opened along the mesenteric border, snap frozen in liquid nitrogen, and stored at −80°C until further use. We found that at day 3 after inoculation, RDEC-H19A-infected rabbits already showed the disease symptoms (liquid stool, lower weight, etc.) compared with those inoculated with the nontoxigenic strain or PBS (33). These changes preceded the ischemia/inflammatory changes detected at day 7 (edema, neutrophil transmigration, etc.).

Immunofluorescence.

Rabbit tissue and T84 cell immunostaining was performed as we previously described (33). Briefly, for Cdc42-GTP immunostaining, 10-μm sections of frozen cecal tissues from rabbits infected with RDEC-H19A bacteria were fixed in 3% formaldehyde in PBS for 30 min, washed extensively in PBS, permeabilized with 0.1% saponin, and blocked with 2% BSA and 15% FBS for 45 min. Sections were incubated with primary antibodies at room temperature for 1 h, washed three times in PBS, and incubated with fluorescently labeled secondary antibodies phalloidin for F-actin and Hoechst for nuclear staining, for an additional 1 h, washed again, immersed in gel mount, and mounted on glass slides for confocal microscopy.

Detection of Stx2 binding sites in human colonic tissue was done as we described (27). Briefly, cryosections of normal human colonic tissue from six individuals were fixed, permeabilized, and blocked as described above. Sections were incubated with Stx2-Alexa568 and B-subunit of cholera toxin (CTB)-Alexa 488 and Hoechst for 1 h, washed again, immersed in gel mount, and covered with coverslips for confocal microscopy.

For cell immunofluorescence experiments, confluent T84 monolayers grown on filters were fixed with 3% formaldehyde in PBS for 10 min, washed extensively in PBS, permeabilized as described above for 30 min, and then incubated with primary and fluorescently labeled secondary antibodies, rinsed, and mounted as described above.

Fluorescence confocal imaging of tissue and cells was performed using Zeiss 510 LSM. Eight- or twelve-bit fluorescence images of confocal optical 0.4-μm sections were collected for further qualitative and quantitative analysis using MetaMorph software (Roper Industries, Marlow, UK).

For SR-SIM images, T84 cells (infected and uninfected) grown and treated as above and labeled with phalloidin-AlexaFluor 488 were mounted on slides using Vectashield mounting medium (Vector Laboratories, Burlingame, CA). The images were obtained using super resolution SIM ELYRA PS.1 system by Zeiss (Jena, Germany).

SEM and TEM imaging.

For scanning electron microscopy (SEM), T84 cells grown on filters were rinsed in 1× PBS for 1 min and then fixed in solution containing 2.0% paraformaldehyde (PFA), 2% glutaraldehyde (GA), 0.1 M sodium cacodylate, 1% sucrose, and 3 mM CaCl₂, pH 7.42, overnight at 4°C. After the buffer rinse, samples were postfixed in 2% osmium tetroxide and 0.1 M sodium cacodylate for 1 h on ice in the dark. After a brief D-H₂O rinse cells were then placed in 2% uranyl acetate for 1 h at room temperature in the dark. After en bloc staining, cells were dehydrated through a graded series of ethanol transferred to a 1:1 mixture of ethanol HMDS (Polysciences) and then passed through two changes of pure HMDS. Drying agent was decanted, and samples were placed in a dessicator with a 15-psi vacuum overnight. Filters were attached to aluminum stubs via carbon sticky tabs (Pella) and coated with 20 nm of AuPd with a Denton Vacuum Desk III sputter coater. Stubs were viewed and digital images captured on a Leo 1530 FESEM operating at 1–3 kV.

For transmission EM (TEM), rabbit cecal tissue samples (~3 mm) were fixed in solution containing 2% GA, 2% PFA, 0.1 M sodium cacodylate, 3 mM CaCl₂, pH 7.2 overnight at 4°C. After the buffer rinse, samples were postfixed in 2% osmium tetroxide in 0.1 M sodium cacodylate for 1 h on ice in the dark. After a brief D-H₂O rinse tissue samples were placed in 2% uranyl acetate for 1 h at room temperature in the dark. After en bloc staining, tissue samples were dehydrated through a graded series of ethanol to 100%, transferred through propylene oxide, embedded in Eponate 12 (Pella), and cured at 60°C for 2 days. Sections were cut on a Riechert Ultracut E with a Diatome Diamond knife. Sections of 80 nm were collected on formvar-coated 1 × 2 mm copper slot grids and stained with uranyl acetate followed by lead citrate. Grids were viewed on a Hitachi 7600 TEM operating at 80 kV and digital images captured with an AMT 1 K × 1 K CCD camera.

EHEC infection causes formation of actin-based macropinocytic blebs at the apical surface of IEC in vitro and in vivo.

We have previously shown that in Gb3 receptor-deficient intestinal epithelial T84 cells, the basal uptake of Stx1 occurs through an actin-dependent mechanism, which we speculated to be MPC (32). MPC is defined as actin-driven, clathrin- and caveolin-independent endocytosis that results in the internalization of fluid and high molecular weight cargo into large irregular vesicles with diameters up to 10 μm (35, 50). The rate of MPC in IEC is generally low but increases significantly upon stimulation by growth factors, protein kinase C, and the nonreceptor tyrosine kinase Src, etc. (12, 24, 36). We have shown that pharmacological stimulation of MPC significantly increased Stx1 endocytosis in T84 cells (33), whereas treatment of T84 cells with cytochalasin D (CytD) significantly inhibited basal Stx1 MPC in T84 cells (32).

We next tested whether the treatment of EHEC-infected T84 cells with 1 μM CytD for 4 h affects Stx1 uptake. Stx1 relative fluorescence intensity in total cell lysates fell from 4.5 ± 0.3 in cells without CytD treatment to 2.4 ± 0.4 in CytD-treated cells (P = 0.01, n = 6), indicating that EHEC-induced Stx1 uptake is an actin-dependent process.

Knowing that EHEC upon attachment and effacement (A/E) to the enterocyte brush border causes a substantial actin rearrangement (6, 10), we speculated that EHEC might induce actin remodeling both locally at sites of A/E and also globally. Thus we have tested the hypothesis that EHEC-induced actin remodeling stimulates MPC, which results in increased Stx1 uptake.

MPC is induced by the activation of plasma membrane-connected actin, which leads to ruffle formation. The macropinocytic ruffles can take different forms, such as lamellipodia, circular ruffles, and, in some cases, large (up to 10 μm) plasma membrane blebs (35). Examination of T84 cells by confocal microscopy showed that EDL933 infection caused both apical and basolateral actin rearrangement and apical actin ruffling (supplemental Fig. S1, A–D). SEM showed substantial rearrangement of the apical surface leading to the disappearance of microvilli and formation of so-called macropinocytic blebs (Fig. 1C). Super resolution structural illumination microscopy (SR-SIM) images confirmed the F-actin nature of these blebs in T84 cells (Fig. 1D). Importantly, the apical blebs appeared in locations that lacked A/E bacteria (Fig. 1C), suggesting that blebs were not a direct consequence of localized bacterial attachment (supplemental Fig. S1E).

To test whether appearance of apical blebs was a characteristic feature of EHEC-induced disease, we used TEM to compare cecal tissue from rabbits infected for 3 days with Stx1-producing RDEC-H19A bacteria to comparable samples from PBS-treated rabbits. RDEC-H19A infection was associated with apical blebbing of cecal epithelial cells (Fig. 1E), morphologically similar to these detected in EHEC-infected T84 cells. Analysis of in vivo model shows that the blebs were formed randomly at the sites without attached bacteria (Fig. 1E) and at some distance from the attached bacteria (Fig. 1F), similarly to our in vitro finding.

Macropinosomes carry Stx1 into and across IEC.

It has been shown (35) that retraction of macropinocytic blebs causes the uptake of Vaccinia virus through formation of actin-coated macropinosomes. We examined Stx1 distribution in EDL933-infected T84 cells and found that toxin was present inside of large actin-coated apical vesicles, which by definition resemble macropinosomes (Fig. 2A).

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Fig. 2.

Stx1 taken up by MPC is transcytosed across the T84 cells in holotoxin form. A: XZ projection through an EHEC-infected T84 cell shows apical F-actin bleb (red) that takes up Stx1 (green). B: XZ projection through EHEC-infected T84 cells with internalized Stx1 shows that Stx1 (green) is transported to the basolateral side of the cells inside the F-actin-coated macropinosomes (red). F-actin, red by phalloidin-AlexaFluor 568; Stx1, green by conjugation to AlexaFluor 488; nuclei, blue by Hoechst. White arrows in A and B indicate the green Stx1-containing vesicles coated with red F-actin. C: SDS-PAGE shows the relative distribution of Stx1A and cleaved Stx1A1 in total cell lysates (endocytosis) and in basal conditioned media (transcytosis) from T84 cells incubated with Stx1 over indicated time in the presence of 50 μM furin inhibitor decanoyl-RVKR-CMK or in EHEC-infected cells. M indicates the corresponding molecular mass marker on a gel. Stx1 taken up by MPC is not cleaved inside the T84 cells 4 h later. Transcytosed toxin collected from basal conditioned media 4 h after EHEC infection is also not cleaved.

We followed the fate of internalized toxin in T84 cells and found that the majority of these actin-coated vesicles did not release the cargo intracellularly over the 4 h of experimental time but rather delivered actin-coated Stx1 to the basal surface of T84 cells (Fig. 2B). To test whether this apical-to-basal Stx1 transport contributes to toxin transcytosis across the T84 monolayers, we measured the relative amount of Stx1 in basolateral conditioned media from infected versus uninfected cells. MPC of Stx1 at the apical surface significantly increased the amount of toxin detected in the basal medium 4 h after infection. Specifically, the presence of apical bacteria resulted in significantly more transcytosed Stx1–680 than when no bacteria were added (Stx1 normalized fluorescence intensity: 8.3 ± 0.5 vs. 3.7 ± 0.4, P < 0.05, n = 12). To further determine whether the transcytosed toxin could have been intracellularly activated, we analyzed the mobility of the Stx form(s) by SDS-PAGE. To generate enzymatically active toxin, the catalytic A-subunit of Stx1 (M_r 32kDa) must be cleaved into two fragments, an active A1 form (M_r 27.5kDa) and a small A2 (M_r 4.5 kDa). Furin, a membrane-associated serine endoprotease, has been shown to activate the toxin in endosomes/trans-Golgi, whereas calpain, a cysteine protease, cleaves Stx1 in the cytoplasm (13, 28). Therefore, detection of the cleaved A1 fragment of Stx1 in total cell lysates or in the basal conditioned media would indicate that internalized Stx had been sorted into an endocytic compartment or released into the cytosol. However, 4 h after EDL933 infection of T84 cells, cellular Stx1 was mostly present in its holotoxin form, with the A1 fragment below detection levels (Fig. 2C), as was the transcytosed Stx1 from basolateral conditioned media. This result suggests that toxin-bearing actin-coated vesicles neither fused with furin-positive compartments nor allowed release of the toxin directly into the cytosol. However, in agreement with published results (43), exposure of uninfected T84 cells to toxin for 24 h resulted in furin-mediated cleavage of intracellular Stx1 to the A1 form, a process that was inhibited by a known furin inhibitor (Fig. 2C).

Taken together, these results demonstrate that early in EDL933 infection, MPC leads to Stx1 uptake and transcellular transcytosis of the holoform, thus providing a route for the holotoxin's systemic dissemination. Clearly, inhibition of EHEC-induced MPC could be an effective means to interfere with toxin-induced systemic diseases. This possibility prompted us to further characterize the molecular mechanism(s) of toxin MPC.

Stx1 uptake by EHEC-induced macropinocytosis is myosin II dependent.

Nonmuscle myosin II (NMII), an actin-based motor protein, has been implicated in MPC that is induced by a variety of stimuli, including viruses, growth factors, etc. in different cell types (9, 22, 35). Therefore, we tested its role in EHEC-induced MPC of Stx1 using reagents that modulate its activity. Blebbistatin is a specific pharmacological inhibitor of NMII ATPase activity (48, 54), and calyculin A at low concentrations specifically inhibits myosin light chain phosphatase (54), thus increasing NMII activity. We found that blebbistatin (50 μM) significantly inhibited EHEC-induced Stx1 uptake (Fig. 3, A and B), whereas calyculin A (5 nM) significantly increased its uptake, indicating that NMII was involved in Stx1 MPC. NMII activity requires phosphorylation of regulatory light chain RLC (34). To confirm that NMII was activated in T84 cells after EDL933 infection, we measured the extent of RLC phosphorylation and found a significant increase (Fig. 3C).

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Fig. 3.

Nonmuscle myosin II (NMII) plays a role in EHEC-stimulated Stx1 MPC. A: representative immunoblot of Stx1-Alexa 680 and GAPDH from infected and uninfected T84 cells that were treated or not with 50 μM blebbistatin or 5 nM calyculin A for 4 h. B: treatments of T84 cells with either 50 μM blebbistatin or 5 nM calyculin A for 4 h affect EHEC-induced Stx1 MPC; n ≥ 3 per each condition. C: immunoblot of T84 total cell lysates shows increase in RLC phosphorylation upon EHEC infection with corresponding quantification of RLC phosphorylation. *Significant compared with uninfected cells (P < 0.05; n = 5). D: representative immunoblot of MNIIA shows the increase in the NMIIA amount in cells infected for 4 h with EDL933 compared with control uninfected cells. E: representative immunoblot of MNIIB shows no changes in the NMIIB amount in cells infected for 4 h with EDL933 compared with control uninfected cells. F–G: NMIIA is present in F-actin apical blebs induced by EDL933. Representative XY optical section through the apical region with corresponding XZ projection of T84 cells uninfected (F) or infected (G) with EDL933 for 4 h and immunostained against NMIIA with F-actin; green by phalloidin-AlexaFluor488 and to visualize NMIIA. The pattern of NMIIA overlaps with the pattern of F-actin at the perimeter of the bleb.

Among three isoforms of NMII (A, B, and C), NMIIA and B but not C are expressed in T84 cells (21). We determined the relative amounts and distributions of both isoforms in control conditions and after 4 h of infection. NMIIA was significantly upregulated after exposure to bacteria (Fig. 3D) but not NMIIB (Fig. 3E). The NMIIA-to-GAPDH ratio in EDL933-infected cells was 2.1 ± 0.6 (P = 0.04, n = 10) when normalized to that of control cells. Furthermore, the intracellular distributions of NMIIA in uninfected versus infected cells were very different. In uninfected cells, NMIIA was exclusively subapical (Fig. 3F and supplemental Fig. S2A), whereas in infected cells it was present throughout the cells (Fig. 3G and supplemental Fig. S2B). Importantly, NMIIA was present all through the apical macropinocytic blebs and concentrated at the perimeter of the blebs where it colocalized with F-actin [yellow color in Fig. 3G (XY plane) and white ring at the bleb perimeter in supplemental Fig. S. 2B (XY plane), which follows the F-actin pattern in the bleb]; 73.1% ± 7.1 of NMIIA colocalized with F-actin in these blebs, whereas only 9.9% ± 2.5 of NMIIA colocalized with the F-actin through the rest of the cells.

The distribution of NMIIB was also altered in EHEC-infected compared with control cells, but this isoform was not present in F-actin blebs (supplemental Fig. S3, A and B). Therefore, it seems that only NMIIA is involved in formation of EHEC-induced actin blebs in T84 cells, and its inhibition significantly decreased Stx1 uptake by EHEC-stimulated MPC.

Cdc42 is necessary but not sufficient for MPC of Stx1.

Macropinosome formation usually requires membrane ruffling. It is well established that Rho GTPases, including Cdc42, are responsible for the triggering of membrane ruffles by activating effectors of actin polymerization, stability, and turnover (15, 56). For example, Cdc42 plays a role in ruffling during Salmonella typhimurium induced MPC in dendritic cells (14). It is also involved in MPC of several types of viruses (35). Consequently, we tested the role of Cdc42 in EHEC-stimulated MPC of Stx1.

EHEC infection led to a significant increase in the amount of Cdc42 in T84 cells after 4 h (supplemental Fig. S4B). Specifically, the Cdc42-to-GAPDH ratio in treated cells was 2.6 ± 0.3-fold increased (P < 0.005, n = 17) over that in control cells, suggesting Cdc42 involvement in Stx1 MPC. To confirm the role of Cdc42 in Stx1 uptake by MPC, we used both pharmacological and molecular approaches. Inhibition of Cdc42 activity by either pirl-1, a specific blocker of nucleotide exchange on Cdc42 (37), or a cell-permeable Cdc42-specific inhibitory peptide (39, 53) significantly decreased Stx1 uptake in EHEC-infected cells (Fig. 4A).

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Fig. 4.

Cdc42 is necessary for EHEC-induced MPC in vitro and in vivo. A: pharmacological inhibition of Cdc42 activity decreases EHEC-stimulated Stx1 uptake in T84 cells; n ≥ 3. NS, nonsignificant. B: Cdc42 KD virtually abolishes the EHEC stimulation of Stx1 uptake. Cdc42KD data represent the combined data from 3 different short hairpin RNA (shRNA) constructs. *Significant compared with control (uninfected T84 cells transduced with lentivirus containing scrambled shRNA; P < 0.05; n = 8). C: constitutively active Cdc42 enhances the EHEC effect on Stx1 endocytosis but is not sufficient to stimulate MPC itself. EV, empty vector; n ≥ 3 for each condition. D: Cdc42 was active (red by anti-Cdc42-GTP Abs, with secondary AlexaFluor 568) in subset of rabbit cecal epithelial cells that often did not have attached bacteria; attached RDEC-H19A bacteria and T84 cell nuclei, blue by Hoechst (33); apical F-actin, green by phalloidin-AlexaFluor 488.

We also employed a knockdown (KD) approach to test the role of Cdc42 in MPC using three different shRNA sequences, as we previously described (Ref. 33 and supplemental Fig. S4A). Cdc42 KD virtually abolished the EHEC-stimulated Stx1 uptake (Fig. 4B). In contrast, cells infected with scrambled shRNA that did not inhibit Cdc42 (Ref. 33 and supplemental Fig. S4A) behaved like a wild type (Fig. 4B). Examination of Cdc42 KD cells by SEM showed a reduction in the number of microvilli (supplemental Fig. S5A), which did not significantly affect bacterial attachment (supplemental Fig. S5B). However, the number and the size of apical blebs in EHEC-infected Cdc42 KD T84 cells (supplemental Fig. S5C) were less, indicating that defects in bleb formation might be responsible for the inhibition of Stx1 uptake in the presence of bacteria.

Conversely, transduction of T84 cells with recombinant adenovirus encoding constitutively active Cdc42 (Q61L) (16) significantly increased Stx1 uptake upon EHEC infection (Fig. 4C). These data show that Cdc42 is necessary for EHEC-stimulated MPC of Stx1. However, constitutively active Cdc42 (Q61L) in the absence of bacteria did not stimulate Stx1 uptake (Fig. 4C), indicating that Cdc42 activation is not sufficient to trigger toxin MPC in T84 cells.

MPC is sensitive to Na/H exchange (NHE) inhibitors, including amiloride, its derivatives, and HOE-694. The mechanism is thought to be due to a decrease in cytosolic pH, which affects both actin assembly and activation of small GTPases, including Cdc42 (26). T84 cells express predominantly two isoforms of plasma membrane Na/H exchanger, basolateral NHE1 and apical NHE2 (4, 17). Inhibition of both by HOE-694 (50 μM) significantly decreased Stx1 endocytosis in EHEC-infected cells (Fig. 4A). This decrease in Stx1 uptake was due to specific inhibition of NHE function, because treatment of T84 cells with 10 μM S3226 (44), a specific inhibitor of the NHE3 isoform, which is not expressed in T84 cells, did not influence EHEC-stimulated Stx1 uptake (Fig. 4A).

Because Cdc42 appears to play an important role in EHEC-stimulated Stx1 uptake in vitro, we tested whether activation of Cdc42 was relevant to the EHEC-induced disease using an animal model. Examination of cecal epithelial tissue from rabbits infected either with Stx1-producing RDEC-H19A strain or with nontoxigenic RDEC-1 bacteria, showed active plasma membrane-bound Cdc42 in the subsets of enterocytes, suggesting that activation of Cdc42 is a molecular event in EHEC infection in vivo (Fig. 4D). Cdc42 plays an important role in EHEC A/E to the apical surface of enterocytes (41, 52). Thus EHEC through type 3 secretion system (T3SS) translocate the effector protein Map to modulate the Cdc42 activity, which is necessary for actin pedestal formation. However, in our in vivo model the active GTP-bound Cdc42 appeared not only in places of bacterial A/E but also in subpopulations of enterocytes having no detectable bacteria nearby (Fig. 4D). This result suggests a potential role for Cdc42 activation in processes beyond the bacterial attachment, including MPC.

We conclude that activation of Cdc42 is a necessary step in the EHEC-stimulated MPC of Stx1, and its inhibition leads to a significant decrease in Stx1 uptake and transcytosis.

Cross-talk between NMII and Cdc42 in EHEC-stimulated macropinocytosis.

NMII controls the activation of small GTPases, including Cdc42, in migrating cells (9, 30). Thus we wondered whether NMIIA activation was upstream of Cdc42 activation in EHEC-stimulated MPC. We tested this possibility by analyzing the effects of inhibitors on either NMII or Cdc42 expression/activation in the presence of EHEC. The Cdc42 inhibitor pirl-1 had no effect on the relative NMIIA amounts either in uninfected cells or in the EDL933-infected cells. Likewise, neither Cdc42 KD nor expression of the constitutively active mutant of Cdc42 changed the EHEC-induced increase in the NMIIA. However, inhibition of NMII activity by blebbistatin significantly impaired the bacteria-induced Cdc42 upregulation (10.5 ± 1.0 in infected cells vs. 6.7 ± 2.4 in infected cells plus blebbistatin, P = 0.03, n = 3). Taken together, these data suggest that Cdc42 works downstream of NMII in EHEC-induced formation of macropinocytic blebs.