INFECTION AND IMMUNITY, Nov. 2005, p. 7113–7125 Vol. 73, No. 110019-9567/05/$08.00�0 doi:10.1128/IAI.73.11.7113–7125.2005Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Cholesterol-Enriched Membrane Microdomains Are Requiredfor Inducing Host Cell Cytoskeleton Rearrangements

in Response to Attaching-Effacing Escherichia coliJason D. Riff,1,2 John W. Callahan,1,3,4 and Philip M. Sherman1,2,3,5*

Research Institute, Hospital for Sick Children,1 and Departments of Laboratory Medicine and Pathobiology,2

Paediatrics,3 and Biochemistry,4 and the Institute of Medical Science,5 University of Toronto,Toronto, Canada

Received 26 January 2005/Returned for modification 18 April 2005/Accepted 8 July 2005

The diarrheal pathogens enterohemorrhagic Escherichia coli (EHEC) O157:H7 strain CL56 and entero-pathogenic Escherichia coli (EPEC) O127:H6 strain E2348/69 adhere intimately to epithelial cells throughattaching-effacing lesions, which are characterized by rearrangements of the host cytoskeleton, intimateadherence, and destruction of microvilli. These cytoskeletal responses require activation of host signal trans-duction pathways. Lipid rafts are signaling microdomains enriched in sphingolipid and cholesterol in theplasma membrane. The effect of perturbing plasma membrane cholesterol on bacterial intimate adherence wasassessed. Infection of both HEp-2 cells and primary skin fibroblasts with strains CL56 and E2348/69 causedcharacteristic rearrangements of the cytoskeleton at sites of bacterial adhesion. CL56- and E2348/69-inducedcytoskeletal rearrangements were inhibited following cholesterol depletion. Addition of exogenous cholesterolto depleted HEp-2 cells restored cholesterol levels and rescued bacterially induced �-actinin mobilization.Quantitative bacterial adherence assays showed that EPEC adherence to HEp-2 cells was dramatically reducedfollowing cholesterol depletion, whereas the adherence of EHEC remained high. Cytoskeletal rearrangementson skin fibroblasts obtained from children with Niemann-Pick type C disease were markedly reduced. Thesefindings indicate that host membrane cholesterol contained in lipid rafts is necessary for the cytoskeletalrearrangements following infection with attaching-effacing Escherichia coli. Differences in initial adherenceindicate divergent roles for host membrane cholesterol in the pathogenesis of EHEC and EPEC infections.

Enterohemorrhagic Escherichia coli (EHEC) serotypeO157:H7 frequently causes outbreaks of bloody diarrhea indeveloped countries, such as occurred in Walkerton, Ontario,Canada, during the summer of 2000, when over 3,000 peoplewere infected and seven deaths occurred (17). Infection withEHEC can be further complicated by the development of thehemolytic uremic syndrome, which is the most common causeof acute renal failure in children (21).

EHEC O157:H7 and the related diarrheal pathogen entero-pathogenic Escherichia coli (EPEC) serotype O127:H6 bothcolonize the host intestinal tract by initial binding eventsfollowed by the development of intimate adhesion throughcharacteristic attaching and effacing lesions. To achieve theattaching-effacing lesion, these bacteria possess a homologouspathogenicity island termed the locus of enterocyte effacement(6, 34) that encodes a type III secretion system. This secretionsystem delivers a number of secreted effector proteins intothe host cell, including EspE, EspB, EspD, EspF, and Map(5, 22, 23).

The eukaryotic plasma membrane is not a homogeneousphospholipid bilayer but contains specialized cholesterol andsphingolipid-rich microdomains, termed lipid rafts (28, 37).Functionally, lipid rafts serve as platforms for protein sortingand membrane trafficking, as well as containing many mole-

cules important for signal transduction events involved in pro-liferation, apoptosis, cell migration, and adhesion (11). In ad-dition, microorganisms and their secreted products utilize lipidrafts in order to exert their effects on host cells (6, 27, 29, 40).The distinct involvement of lipid rafts in signaling functions(10) led us to hypothesize a role for these cholesterol-enrichedmicrodomains in the formation of E. coli-induced attaching-effacing lesions. In this study, the involvement of host cell lipidrafts in the formation of attaching-effacing lesions was deter-mined using complementary approaches. The results demon-strate that host plasma membrane cholesterol is required forbacterial adherence and attaching-effacing cytoskeleton alter-ations in response to both EHEC O157:H7 and EPECO127:H6 infections.

MATERIALS AND METHODS

Tissue culture and cell lines. HEp-2 human laryngeal epithelial cell line (CCL23; American Type Culture Collection, Manassas, VA), were cultured at 37°Cand 5% CO2 in minimal essential medium supplemented with 10% fetal bovineserum, 1% sodium bicarbonate, 1% Fungizone, and 1% penicillin-streptomycin(all media and supplements from Life Technologies, Grand Island, NY).

Patients with Niemann-Pick type C (NPC) disease (Hospital for Sick Children)were defined using the cholesterol esterification assay as previously described(2). Fibroblasts were grown in at 37°C and 5% CO2 in �-minimal essentialmedium (Wisent Inc., Saint-Jean-Baptiste de Rouville, Canada) supplementedwith 10% fetal bovine serum (Life Technologies).

Bacterial growth and conditions of infection. EHEC serotype O157:H7, strainCL56, and EPEC serotype O127:H6, strain E2348/69, were held on 5% sheepblood agar plates at 4°C. Individual colonies were scraped into Penassay broth(Difco Laboratories, Detroit, Mich.) and grown for 18 h at 37°C before use inexperimental infection, as previously described (4). For experimental infection,stationary-phase bacteria were added to tissue culture cells grown in Lab-Tek

* Corresponding author. Mailing address: Room 8409, Hospital forSick Children, 555 University Avenue, Toronto, Ontario M5G 1X8,Canada. Phone: (416) 813-7734. Fax: (416) 813-6531. E-mail:sherman@sickkids.ca.

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four-well chamber slides (Nalge Nunc International, Naperville, IL) or 10-cm-diameter tissue culture dishes (Starstedt Inc., Montreal, Canada) at a multiplicityof infection of 100 bacteria to 1 eukaryotic cell, for 3 to 6 h at 37°C in antibiotic-free tissue culture medium. The cells were then washed six times with phosphate-buffered saline (PBS) to remove nonadherent bacteria and processed further asdescribed below.

Cholesterol perturbation. Methyl-�-cyclodextrin (M�CD; Sigma ChemicalCo., St. Louis, MO) was employed to remove cholesterol from the plasmamembrane and disrupt the function of lipid rafts in eukaryotic cells (24). Prior tobacterial infection, HEp-2 cells were incubated with 1, 3, or 10 mM M�CD inantibiotic-free medium for 1 h at 37°C. The medium was aspirated, and the cellswere washed with PBS to remove solubilized cholesterol and remaining M�CD.To add cholesterol back into cholesterol-depleted HEp-2 cells, 20, 100, or

200 �g/ml soluble cholesterol (cholesterol complexes with M�CD; Sigma) inantibiotic-free medium was added to cells for 45 min at 37°C. Following deple-tion/repletion of cholesterol, the cells were washed with PBS before bacterialinfection was continued. In another set of experiments, HEp-2 cells were treatedwith filipin complex (Sigma) in order to determine the effect of cholesterolsequestration on E. coli infection. HEp-2 cells were incubated with 0.1, 1, and5 �g/ml filipin for 1 h at 37°C prior to infection and during infection with EHECand EPEC.

Thin-layer chromatography. Confluent HEp-2 cells grown in 75-cm2 flasks(approximately 6 � 107 cells per flask) were either left untreated, depleted withM�CD, or replenished with cholesterol-M�CD complexes. The cells were thenlifted from the flask surface with 0.05% trypsin (Life Technologies), pelleted, andwashed twice with PBS. Cells were resuspended in 20-ml glass tubes, and cellular

FIG. 1. EHEC O157:H7-induced cytoskeletal rearrangements are disrupted by cholesterol depletion. Immunofluorescence micrographs (a toc) using an �-actinin antibody (left column) and an EHEC O157:H7 antibody (middle column) and merged images (right column). (a) Uninfectedcells show no cytoskeletal rearrangements and lack bacterial staining. (b) Cells infected with EHEC O157:H7 showed foci of bacterial adherenceat sites of EHEC O157:H7-induced �-actinin mobilization (arrows). (c) HEp-2 cells depleted of cholesterol (10 mM M�CD; 1 h) prior to EHECO157:H7 infection did not demonstrate focal cytoskeletal rearrangements at sites of diffusely adherent bacteria. (d) Graphical representation ofsemiquantification of cytoskeletal rearrangements with increasing concentrations of M�CD. The findings show a dose-dependent decrease in theformation of EHEC O157:H7-induced �-actinin foci (ANOVA, P � 0.001). The error bars indicate standard errors.

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lipids were extracted by incubating them in a 2:1 (vol/vol) chloroform-methanolsolution overnight at room temperature with gentle shaking. After being filteredto remove precipitated proteins, the cleared solution was subjected to Folchextraction (9). Briefly, distilled water was added to obtain a solution of chloro-form-methanol-water (2:1:0.6 [vol/vol/vol]). The tubes were briefly agitated andthen allowed to stand at room temperature overnight for phase separation. Thelower organic phase, containing cellular lipids, was then aspirated and driedunder nitrogen gas. Samples were resuspended in 0.1 ml 2:1 chloroform-meth-anol, and a 20-�l sample was dotted onto a thin-layer chromatogram plate. Freecholesterol (50 �g; Sigma) was employed as the reference standard. A 70:30:1(vol/vol/vol) hexane-diethyl ether-acetic acid developing solution was used toseparate the lipids, and the dried plates were stained with iodine vapor tovisualize bands corresponding to cholesterol.

Cell viability. The LIVE/DEAD Viability/Cytotoxicity assay (MolecularProbes Inc., Eugene, OR) was used to determine the viability of adherent HEp-2cells after treatment with M�CD. HEp-2 cells were seeded onto 22- by 22-mmglass coverslips (VWR Scientific Inc., Media, PA) in multiwell plates at a con-centration of 105 cells/well and allowed to adhere overnight at 37°C in 5% CO2.The cells were then either left untreated or depleted of cholesterol, as describedabove. Coverslips were washed twice with PBS and, according to the manufac-turer’s instructions (Molecular Probes), incubated with 2 ml of 1 �M calcein AMand 2 �M ethidium homodimer 1 in PBS for 40 min at room temperature.The cells were visualized using a Leitz Dialux 22 microscope (Leica Canada,Willowdale, Ontario, Canada) at �100 magnification. Four random fields percoverslip were counted by direct visualization.

Immunofluorescence microscopy. HEp-2 cells and human skin fibroblasts wereseeded onto Lab-Tek four-well slides (Nalge Nunc) at an approximate density of105 cells/well and allowed to adhere overnight in 5% CO2 at 37°C. The cells werethen washed twice with PBS and transferred into serum-free medium. Afterinfection for 3 to 4 h at 37°C and 5% CO2 and subsequent removal of nonad-herent bacteria, the cells were fixed in 100% methanol for 10 min at 4°C.Labeling of the cell cytoskeleton and bacterial antigens by immunostaining wasemployed to clearly identify the colocalization of attaching-effacing lesions andbacteria. Cytoskeleton rearrangements were detected using anti-�-actinin mouseimmunoglobulin M (IgM) (Sigma) and fluorescein isothiocyanate-labeled don-key anti-mouse IgM (Jackson Immunoresearch Laboratories Inc., West Grove,PA) as described previously (19). In some experiments, cells were probed for the

localization of caveolae with an anti-caveolin-1 antibody (Santa Cruz Biotech-nology Inc., Santa Cruz, CA) and rhodamine-conjugated donkey anti-rabbit IgG(Jackson Immunoresearch). Adherent bacteria were visualized with anti-Escherichia coli O157:H7 polyclonal goat antibody (Kirkegaard & Perry Labo-ratories Inc., Gaithersberg, MD) and rhodamine-conjugated donkey anti-goatantibody (Jackson Immunoresearch).

Binding of EPEC to cells was detected using rabbit serum obtained fromanimals immunized with heat-killed EPEC O127:H6 suspended in Freund’scomplete adjuvant and rhodamine-conjugated donkey anti-rabbit IgG (JacksonImmunoresearch). Primary and secondary antibodies were both diluted 1:100 insterile PBS and added separately for 1 h at 37°C or room temperature, respec-tively. The wells were washed six times with PBS between incubations. Vecta-shield (Vector Laboratories Inc., Burlingame, CA) mounting medium was thenadded, and slides were mounted with 22- by 50-mm glass coverslips (VWR) andsealed before being viewed. Samples were viewed under a Leitz Dialux 22fluorescence microscope (Leica Canada) at �200 and �400 magnifications.

Electron microscopy. For scanning electron microscopy, HEp-2 cells wereseeded onto 22- by 22-mm glass coverslips (VWR) in multiwell plates at105 cells/well. The cells were allowed to adhere overnight, washed twice withPBS, and then transferred to antibiotic-free medium. The cells were either leftuntreated or depleted of cholesterol with 10 mM M�CD (1 h at 37°C and 5%CO2) and either left uninfected or infected with EHEC O157:H7 as describedabove. After infection, the wells were washed six times with PBS and fixed inuniversal fixative (4% paraformaldehyde, 1% glutaldehyde in 0.1 M phosphatebuffer) for at least 1 h. The coverslips were then removed from the multiwellplates and incubated in 2% osmium tetroxide for 1 h at room temperature. Thecells were then dehydrated in a graded series of ethanol (50% to 100%), driedthrough a critical-point dryer, and sputter coated with gold. Samples were viewedwith a JSM 820 (Joel USA Corp., Peabody, MA) scanning electron microscope.

For transmission electron microscopy, HEp-2 cells were grown in 6-cm-diam-eter tissue culture dishes (Becton Dickinson and Co., Franklin Lakes, NJ) untilconfluent. The cells were then left untreated or treated with M�CD (1 to 10 mMfor 1 h at 37°C) and subsequently infected with EHEC, as described above. Afterbeing washed six times in PBS to remove nonadherent bacteria, the monolayerswere fixed with 2.5% glutaldehyde in 0.1 M phosphate buffer, pH 7.4, for 10 min.HEp-2 cells were scraped from the tissue culture dishes and pelleted at 600 rpmin the fixative buffer. The HEp-2 cell pellets were next postfixed in 2% aqueousosmium tetroxide for 1 h. Dehydration was then performed in graded acetone,followed by embedding in epoxy resin. Osmium fixation, dehydration, and em-bedding were conducted in a Pelco Biowave microwave oven (Pelco Interna-tional, Redding, CA) similar to the procedure described by Giberson et al. (14).One-micrometer-thick sections were stained with toluidine blue, and ultrathinsections were stained with uranyl acetate and lead citrate. Transmission electronmicroscopy examination was performed under a JEM 1230 (Joel USA Corp.,Peabody, MA) transmission electron microscope.

Quantification of bacterial adherence. CFU counts were performed to deter-mine the effect of cholesterol depletion on initial bacterial attachment to tissueculture cells. HEp-2 cells were grown in 10-cm-diameter tissue culture dishes(Starstedt) until confluent. The cells were then left untreated or treated withM�CD (1 to 10 mM for 1 h at 37°C) and subsequently infected with EHEC orEPEC, as described above. After being washed six times in PBS to removenonadherent bacteria, HEp-2 cells with adherent bacteria were lysed in distilledwater for 5 min at room temperature. Bacteria were then serially diluted in PBSand plated onto McConkey agar, and CFU counts were calculated after over-night growth at 37°C to determine the number of viable bacteria adherent to thetissue culture cells.

Semiquantification of bacterially induced cytoskeletal rearrangements. Cy-toskeletal-rearrangement events were quantified by visual counting from immu-nofluorescence photomicrographs. Greater than 100 cells in four random fieldscontaining at least 25 HEp-2 cells stained for �-actinin were quantified per well.The results are expressed as the average number of �-actinin foci � standarderror per HEp-2 cell in four separate experiments.

Statistical analysis. Quantitative and semiquantitative results are expressed asmeans � standard errors. Statistical significance was determined by analysis ofvariance (ANOVA), followed by the Tukey-Kramer multiple-comparison test.

RESULTS

HEp-2 cell cholesterol, but not viability, is altered by treat-ment with M�CD. To determine if M�CD treatment couldaffect HEp-2 cell cholesterol levels, lipid extracts of untreated,depleted, and depleted/replenished cells were analyzed by

FIG. 1—Continued.

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thin-layer chromatography (data not shown). Cholesterol de-pletion was dose-dependent, since treatment of HEp-2 cellswith 1 mM and 3 mM M�CD resulted in a slight decrease incholesterol content, whereas treatment with 10 mM M�CDdramatically decreased HEp-2 cholesterol content comparedwith untreated HEp-2 cells. Addition of soluble cholesterol(20 �g/ml, 100 �g/ml, and 200 �g/ml) increased the HEp-2 cellcholesterol level from the depleted state. The percentage ofnonviable cells in treated samples showed only modest increaseafter treatment with 1 mM M�CD (3.8% � 0.8%), 3 mMM�CD (3.3% � 0.7%), and 10 mM M�CD (9.7% � 1.6%) incomparison with the untreated control (1.6% � 0.5%).

Cholesterol depletion of HEp-2 cells inhibits cytoskeletalrearrangements induced by both EHEC and EPEC. As shownin Fig. 1a, in the absence of bacteria, the cytoskeletal protein�-actinin was distributed throughout the eukaryotic cells in auniform manner. EHEC O157:H7 infection of HEp-2 cellscaused characteristic cytoskeletal rearrangements visible asdense foci of �-actinin present directly underneath adherentbacteria (Fig. 1b). At sites of �-actinin mobilization, there wascolocalization of bacteria and cytoskeletal components, asshown by the presence of both �-actinin and bacterial stainingin the merged image (Fig. 1b). In contrast, HEp-2 cells de-pleted of cholesterol with M�CD (10 mM; 1 h) showed a

FIG. 2. EPEC-induced cytoskeletal rearrangements are disrupted by cholesterol depletion. Immunofluorescence micrographs (a to c) using an�-actinin antibody (left column) and EPEC immune serum (middle column) and merged images (right column). (a) Uninfected cells show nocytoskeletal rearrangements and lack bacterial staining. (b) Cells infected with EPEC showed microcolony-type bacterial adherence at sites ofEPEC-induced �-actinin mobilization (arrows). (c) HEp-2 cells depleted of cholesterol (10 mM M�CD; 1 h) prior to EPEC infection show areduced number of focal cytoskeletal rearrangements at the few sites of bacterial adherence. (d) Graphical representation of semiquantificationof cytoskeleton rearrangements with increasing concentrations of M�CD. The findings show a dose-dependent decrease in the formation ofEPEC-induced �-actinin foci (ANOVA, P � 0.001). The error bars indicate standard errors.

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reduced number of cytoskeletal-rearrangement events beneathsites of bacterial adhesion (Fig. 1c). The inhibitory effect wasquantified by counting the number of dense �-actinin focidisplayed by infected HEp-2 cells from photomicrographs. Asshown in Fig. 1d, M�CD inhibited the ability of EHECO157:H7 to induce host cell cytoskeletal rearrangements in adose-dependent manner.

Similar results were observed following infection of HEp-2cells with the prototype EPEC strain, E2348/69. Photomicro-graphs of HEp-2 cells infected with EPEC O127:H6 displayedcharacteristic microcolony formation and �-actinin rearrange-ments at sites of bacterial adherence (Fig. 2b). Formation ofmicrocolonies and rearrangement of �-actinin (Fig. 2c) wasreduced in cells depleted of cholesterol by M�CD in a dose-dependent manner (Fig. 2d).

Adhesion of EPEC, but not EHEC, to HEp-2 cells is reducedby cholesterol depletion. Fewer bacteria were adherent toM�CD-treated HEp-2 cells following EPEC infection (Fig. 2c)than to untreated cells (Fig. 2c). To confirm this observation,monolayers of HEp-2 cells treated with various doses ofM�CD (1, 3, and 10 mM; 1 h) were infected with either EHECor EPEC, and the total number of bacteria bound to themonolayer was assessed by determining the bacterial CFU. Asshown in Table 1, the number of EHEC bacteria bound tocholesterol-depleted HEp-2 monolayers remained high (7.64log10 adherent EHEC bacteria bound to 10 mM M�CD-treated monolayers versus 7.87 log10 bound to untreatedmonolayers). In contrast, EPEC adherence was reduced toapproximately 14% of bacterial adherence observed in un-treated HEp-2 monolayers (6.46 log10 adherent EPEC bacteria

bound to 10 mM M�CD-treated monolayers versus 7.34 log10

bound to untreated monolayers).Replenishment of HEp-2 cells with soluble cholesterol res-

cues EHEC and EPEC patterns of localized adherence. Toconfirm that inhibition of �-actinin mobilization was attribut-able to cholesterol removal, cholesterol-depleted HEp-2 cellswere replenished with exogenous cholesterol. ReplenishingHEp-2 cells with 200 �g/ml soluble cholesterol prior to bacte-rial infection rescued the ability of these bacteria to recruitcytoskeletal components to sites of bacterial adhesion (Fig. 3)to levels comparable to those observed in the positive control.

Disruption of caveolae by cholesterol sequestration does notaffect attaching-effacing cytoskeleton rearrangements in hostcells. The cholesterol-sequestering agent filipin is commonlyused to disrupt lipid rafts and caveolae (32, 39, 41), anothercholesterol-dependent membrane microdomain involved inprotein sorting and membrane trafficking. Unlike M�CD, fili-pin does not remove cholesterol from the plasma membranebut rather binds to cholesterol within the membrane. Treat-ment of HEp-2 cells with filipin (0.1 to 5 �g/ml for 60 min priorto and during infection at 37°C) did not prevent EHEC(Fig. 4a)- or EPEC (Fig. 4b)-induced �-actinin recruitment.Furthermore, HEp-2 cells did not show caveolin 1 recruitmentto sites of bacterial adhesion and �-actinin mobilization(Fig. 4c), suggesting that intact plasma membrane lipid rafts,and not caveolae, are necessary for EHEC- and EPEC-inducedcytoskeleton perturbations.

Cholesterol depletion prevents effacement of HEp-2 cell sur-face structures. In addition to cytoskeletal protein rearrange-ments, bacterial intimate adherence also causes effacement ofmicrovilli on the surfaces of intestinal epithelial cells in vivo(21). The surface of an untreated HEp-2 cell is covered withsurface appendages, as visualized by scanning electron micros-copy (Fig. 5a). Following EHEC O157:H7 infection, thesestructures were effaced, revealing a smooth cell surface(Fig. 5b). Cholesterol depletion (10 mM M�CD for 1 h at37°C) alone led to a shortening of appendage length (Fig. 5c).EHEC O157:H7-infected HEp-2 cells that were pretreatedwith M�CD (Fig. 5d) retained these shortened structures, re-sembling the surface of an uninfected cholesterol-depleted cell.Furthermore, replenishment of HEp-2 cells with 200 �g/ml sol-uble cholesterol restored the surface appendages (Fig. 5e) andrescued the ability of EHEC O157:H7 to efface the surfaces ofHEp-2 cells (Fig. 5f).

Cholesterol depletion prevents the formation of actin-richpedestals beneath adherent bacteria. As a complementary ap-proach to further characterize the inhibitory action of host cell

FIG. 2—Continued.

TABLE 1. Adhesion of EHEC and EPEC to HEp-2 cellmonolayers after cholesterol depletion

M�CD (mM)No. of adherent bacteriaa

EHEC EPEC

0 7.87 � 0.03 7.34 � 0.061 7.85 � 0.05 7.27 � 0.063 7.79 � 0.05 6.75 � 0.07c

10 7.64 � 0.04b 6.46 � 0.09c

a Values are log10 means � standard error (n � 3 duplicate samples).b P � 0.01.c P � 0.001.

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cholesterol depletion on EHEC adhesion, transmission elec-tron microscopy was undertaken. Figure 6a shows a cross-sectional view of the plasma membrane region of an uninfectedand untreated HEp-2 cell. At high magnification, an EHEC-infected HEp-2 cell (Fig. 6b) displays distinct actin-rich cups atsites of bacterial intimate adherence and no evidence of sur-face appendages. In contrast, HEp-2 cells treated with 10 mMM�CD for 1 h prior to EHEC infection (Fig. 6d) displaysurface appendages and no actin-rich cups at sites of bacterialadherence. Addition of exogenous cholesterol (200 �g/ml sol-uble cholesterol; 45 min) restored EHEC-induced actin-rich-

cup formation (Fig. 6f) characteristic of attaching-effacinglesions.

Niemann-Pick type C fibroblasts are resistant to attaching-effacing E. coli-induced cytoskeleton rearrangements. Skinfibroblasts from human patients with NPC disease are charac-terized by a cholesterol-trafficking defect whereby cholesterol-enriched microdomains are reduced at the plasma membrane(12). Fibroblasts from NPC patients were analyzed for choles-terol ester (CE) synthesis to confirm their phenotype (2). TwoNPC cell lines, 15055 and 16934, showed decreased CE syn-thesis (1.6 � 1.3 and 4.1 � 0.5 nmol/24 h/mg protein, respec-

FIG. 3. The attaching-effacing phenotype is rescued by the addition of exogenous cholesterol to HEp-2 cells. Immunofluorescence micrographsusing an �-actinin antibody (left column) and EPEC immune serum (middle column) and merged images (right column). (a) Cells infected withEPEC show the adherence of microcolonies at sites of cytoskeletal rearrangement typical of EPEC-induced attaching-effacing lesions. (b) HEp-2cells depleted of cholesterol prior to EPEC infection do not possess focal cytoskeletal rearrangements at the few sites of bacterial adherence. (c)HEp-2 cells depleted of cholesterol were replenished and display cytoskeletal rearrangements (arrows), as well as EPEC microcolony adherencepatterns comparable to the untreated control. Comparable results were obtained with EHEC O157:H7-infected HEp-2 cells.

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tively) compared to two control cell lines from non-NPC pa-tients, 4993 and 15215, which showed high CE synthesis(38.4 � 6.2 and 54.8 � 2.2 nmol/24 h/mg protein, respectively).Cytoskeletal rearrangements were induced by both EHEC(Fig. 7b) and EPEC (Fig. 7c) on control primary fibroblasts. Bycontrast, EHEC (Fig. 7e) and EPEC (Fig. 7f) did not inducefoci of cytoskeletal components beneath adherent bacteria onNPC fibroblasts.

DISCUSSION

In this study, the critical role of host cell plasma membranecholesterol in bacterial adhesion and attaching-effacing lesionformation in response to the diarrheal pathogens entero-hemorrhagic E. coli O157:H7 and enteropathogenic E. coliO127:H6 has been demonstrated for the first time. Treatmentof HEp-2 cells with M�CD removed cholesterol, did not affect

FIG. 4. Bacterially induced cytoskeleton mobilization events are caveola independent. (a and b) Immunofluorescence micrographs stained for�-actinin, EHEC, or EPEC and merged images. HEp-2 cells were treated with 1 mg/ml filipin for 1 h prior to infection and during infection at37°C. Filipin treatment did not prevent EHEC O157:H7-induced (a) or EPEC-induced (b) cytoskeletal rearrangements. (c) Immunofluorescencemicrographs stained for �-actinin and caveolin 1 and light microscopy photomicrographs show bacterial adherence to HEp-2 cells. EHECO157:H7-induced cytoskeletal rearrangements (arrows) did not display recruitment of caveolin 1 (middle) to sites of bacterial adhesion (right).Comparable results were seen during EPEC infection.

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FIG. 5. EHEC O157:H7-induced HEp-2 cell surface effacement is prevented following cholesterol depletion. Scanning electron photomicrographsof uninfected (left column) and EHEC O157:H7-infected (right column) HEp-2 cells. (a) Untreated HEp-2 cells display appendages covering the surfaceof the cell, while EHEC O157:H7-infected cells (b) demonstrate a smooth cell surface. HEp-2 cells depleted of cholesterol possess shortened surfacestructures covering both (c) uninfected and (d) EHEC-infected cells. HEp-2 cells depleted of cholesterol using M�CD and then replenished withexogenous cholesterol (e) displayed structures approaching those of untreated cells, which are effaced following EHEC adhesion (f). Approximatemagnification, �6,000.

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FIG. 6. EHEC O157:H7-induced actin pedestal formation is prevented following cholesterol depletion. Transmission electron photomicrographs ofHEp-2 cells (a and b) untreated, (c and d) cholesterol depleted (10 mM M�CD; 1 h), and (e and f) replenished with exogenous cholesterol (200 mg/ml;45 min). HEp-2 cells infected with EHEC O157:H7 (b) possess distinct actin-rich cup-like pedestals (arrows) underlying intimately adherent bacteria. Bycontrast, dense actin-rich cups are not present at the sites of EHEC adhesion to HEp-2 cells depleted of cholesterol (d). Replenishment of HEp-2 cellspreviously depleted of cholesterol rescues the ability of EHEC to recruit dense foci of actin (arrows). Bar, 500 nm.

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host cell viability, and yet prevented bacterially induced rear-rangement of the host cytoskeleton. This effect was dose de-pendent and reversible by reintroducing cholesterol into de-pleted HEp-2 cells. Cholesterol depletion of HEp-2 cellsdid not affect the initial adhesion of EHEC to HEp-2 cells,whereas EPEC adherence was reduced. This indicates thatmembrane cholesterol may differentially regulate the bindingof EHEC and EPEC to host cell surfaces.

While removal of cholesterol by M�CD showed inhibitoryeffects, filipin did not prevent EHEC O157:H7-induced cytoskel-eton rearrangements, nor was caveolin 1 recruited to these sites ofbacterial adhesion. This demonstrates that the caveolar subclassof lipid rafts is not involved in attaching-effacing pathogenesis.Although lipid raft-associated components are reported to local-ize to sites of EPEC binding (16, 42), this is the first reportdemonstrating that removal of cholesterol, a required structuralelement of lipid rafts (41), from host cells inhibits attaching-effacing lesion formation. The necessity for cholesterol was con-firmed using a naturally occurring mutant human cell with de-creased lipid raft cholesterol (12). Taken together, these resultsindicate that intact sphingolipid/cholesterol-enriched microdo-mains are required for attaching-effacing lesion formation in re-sponse to both EHEC and EPEC infections.

In the present study, while the number of EHEC- andEPEC-induced �-actinin foci was reduced following choles-terol depletion of host cells, EPEC, but not EHEC, adhesionto HEp-2 cells was decreased. These findings indicate thatwhile only intimate adherence of EHEC O157:H7 is regulatedby cholesterol, cholesterol may function in the regulation ofboth initial binding and intimate adhesion of EPEC. Preciselyhow cholesterol mediates adhesion of EPEC is not known.While EPEC does not directly bind to cholesterol (1), manip-ulation of the cholesterol concentration in cells could indirectlyaffect lipid raft localization of an EPEC receptor. Such spec-ulation is not without precedent, since studies of the oxytocinreceptor have shown that binding of oxytocin is dependent onmembrane interactions of the receptor with cholesterol in lipidrafts (15). Alternatively, cholesterol depletion could result inthe efflux of other lipids, such as phosphatidylethanolamine, alipid that has been shown to be a receptor of EPEC adherence(1) which is released after cholesterol depletion (33). On theother hand, EPEC forms characteristic microcolonies medi-ated by interbacterial binding of the bundle-forming pilus (21,26). A decrease in EPEC microcolony formation on cholester-ol-depleted HEp-2 cells could be responsible for the reducedadherence observed.

The cholesterol depletion agent M�CD has been used inother signal transduction-dependent systems to study the ef-

fects of cholesterol removal on lipid raft function, includingepidermal growth factor receptor (18) and T-cell receptor sig-naling (31). It has recently been appreciated that lipid raftfunction is important for microbial pathogens, including hu-man immunodeficiency virus infection of CD4� T cells (38)and Mycobacterium tuberculosis infection of macrophages (13),as well as bacterial toxins, such as cholera toxin (29) andvacuolating cytotoxin A (34).

M�CD-mediated cholesterol efflux has been described ashighly specific compared to other cyclodextrins (24), but it mayalso induce efflux of other membrane lipids, including glyco-sphingolipids, as well as induce cytotoxicity (33). Since previ-ous studies have demonstrated variable effects on the viabilityof cells after depletion of cholesterol by M�CD, the viability ofM�CD-treated HEp-2 cells was compared to that of untreatedcontrols. As cholesterol depletion of epithelial cells was revers-ible by addition of exogenous cholesterol without inducingcytotoxicity, it is reasonable to conclude that the inhibitoryeffects on host cytoskeleton recruitment are specifically due tothe removal of cholesterol.

Signal transduction responses and remodeling of the cy-toskeleton in host epithelial cells are both required for forma-tion of the attaching-effacing lesion. Dense foci of cytoskeletalproteins, including F-actin, �-actinin, talin, and ezrin, aggre-gate immediately beneath adherent EHEC and EPEC (16).Activation of signaling pathways is involved in reorganizationof the cytoskeleton, including phosphatidylinositol 3 kinaseand phospholipase C-gamma (19). Membrane-associatedEHEC and EPEC effectors, such as EspE/Tir and EspB, mayassociate with lipid rafts, either directly or indirectly, to exertlocal effects at sites of bacterial adhesion. Pathogenic bacteriacommonly inject type III secreted proteins into host cells tousurp or disrupt signaling pathways to induce cytoskeleton-dependent internalization of bacteria (3) or prevent phagocy-tosis by macrophages (20). Many of the signaling moleculesaffected, including small G proteins, such as Rac and Cdc42,and phosphatidylinositides, are lipid raft dependent (8). Sal-monella enterica type III effectors, PipB and PipB2, are en-riched in detergent-resistant microdomains (25).

As a complementary approach to depletion of cholesterolwith M�CD, primary human skin fibroblasts from patients withNPC disease were infected with EHEC and EPEC and as-sessed for attaching-effacing lesion formation. NPC is a rare,heritable, and fatal neurodegenerative disorder affecting hu-mans (35). Mutations of NPC1 or NPC2 lead to a dysfunctionin intracellular cholesterol trafficking, resulting in high internalcholesterol levels retained within lysosomal compartments anddecreased cholesterol in the trans-Golgi network (30). Al-

FIG. 7. Cytoskeletal rearrangements formed in response to EHEC and EPEC infections are reduced on Niemann-Pick type C primary humanskin fibroblasts compared with wild-type fibroblasts. Immunofluorescence micrographs using an �-actinin antibody (left column) and bacterialantiserum (middle column) and merged images (right column). Results using fibroblasts from an unaffected individual (cell line 4993) are shownin panels a to c. Results using fibroblasts derived from a patient with NPC (cell line 16934) are presented in panels d to f. (a) Uninfected cells showno cytoskeletal rearrangements and lack staining with primary antibodies against bacteria. (b) Cells infected with EHEC show bacterial adherenceat sites of cytoskeletal rearrangement typical of EHEC-induced attaching-effacing lesions (arrows). (c) Cells infected with EPEC also demonstratedmicrocolonies adherent at sites of cytoskeletal rearrangement characteristic of EPEC-induced attaching-effacing lesions. (d) Uninfected NPC cellsshow no cytoskeletal rearrangements and lack bacterial staining. NPC fibroblasts infected with EHEC (e) or EPEC (f) do not possess foci ofcytoskeleton proteins at sites of bacterial adhesion. Comparable results were observed using two additional skin fibroblast cell lines (wild-type15215 and NPC cell line 15055).

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though the total plasma membrane cholesterol contents innpc1/ cells are similar to those in wild-type cells, the cho-lesterol contents of lipid rafts isolated from npc1/ cells aremarkedly reduced (12). Thus, npc1/ cells are an effectivetool to study lipid raft-dependent processes. Both EHEC andEPEC were attenuated in their ability to recruit foci of �-ac-tinin to sites of bacterial adhesion on npc1/ fibroblasts incomparison with control cells. This observation also indicatesthat intact plasma membrane cholesterol-enriched microdo-mains are required for EHEC and EPEC intimate attachment.

In summary, this study has shown that perturbation ofplasma membrane cholesterol in host cells reduces EHEC- andEPEC-induced cytoskeletal alterations. Since bacterial adher-ence and attaching-effacing lesion formation are important foreliciting human disease, this study highlights the role of cho-lesterol-enriched microdomains in the pathobiology of disease.Developing a more precise understanding of the molecularmechanisms underlying EHEC and EPEC infections shouldaid in the development of novel intervention strategies.

ACKNOWLEDGMENTS

We thank Clifford Lingwood and Anita Nutikka for assistance withthin-layer chromatography procedures, Nancy Cracknell for assistancewith primary fibroblast culturing procedures, and Yew Meng Heng andJulia Huang for assistance with electron microscopy. We also thankDanny Aguilar for assistance with preparation of the figures and PeterCeponis for critical review of the manuscript.

This work was supported by an operating grant from the CanadianInstitute for Health Research. J.D.R. is supported through a student-ship by the Ontario Student Opportunity Trust Fund-Hospital for SickChildren Foundation Student Scholarship Program and a University ofToronto Fellowship. P.M.S. is the recipient of a Canada ResearchChair in Gastrointestinal Disease.

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Editor: V. J. DiRita

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