Invasion of enteropathogenic Escherichia coli into host cells through epithelial tight junctions Qiurong Li 1,2 , Qiang Zhang 1,2 , Chenyang Wang 1 , Ning Li 1 and Jieshou Li 1 1 Institute of General Surgery, Jinling Hospital, Nanjing, China 2 School of Medicine, Nanjing University, Nanjing, China As pathogens invade the gastrointestinal tracts, they must overcome epithelial barriers to initiate infection. An intact epithelial barrier is essential for physiological homeostasis and defense against extrinsic antigens. The intestinal barrier comprises an intact layer of epithelial cells, which are tightly connected in the apical region of the lateral plasma membrane by specialized narrow belt-like structures called tight junctions (TJs). TJs are dynamic structures composed of various proteins, including the transmembrane proteins occludin, a fam- ily of claudins, and ZO-1, a peripheral membrane pro- tein of TJs [1]. Disruption of tight-junction integrity is a mechanism by which diarrhea is induced, and TJs are a common target of various enteric pathogens [2]. The human intestinal pathogen enteropathogenic Escherichia coli (EPEC) causes diarrheal disease by dis- rupting the integrity of TJs. EPEC disrupts TJ archi- tecture by loss of TJ protein–protein interactions, redistribution of TJ proteins, and the appearance of aberrant TJ strands in the lateral membrane [3]. Recently, TJs have been considered as specialized plasma membrane microdomains, lipid raft-like mem- brane compartments that are enriched in cholesterol and sphingolipid [4]. It was suggested that these raft- like compartments play an important role in the spatial organization of TJs and probably in regulation of paracellular permeability in epithelial cells [4]. The loss of TJ barrier function has been correlated with displacement of lipid raft-associated TJ proteins, suggesting that this membrane microdomain is an integral part of the TJ structure [4]. The critical role of the host cell plasma membrane in response to pathogens has been demonstrated by studies indicating that lipid rafts are the preferred entry sites for several invasive pathogens including Salmonella, Shigella, Listeria and Chlamydia [5–8]. It is likely that Campylobacter jejuni also utilizes lipid rafts as the entry point [9]. Coxsackievirus, adenoviruses Keywords enteropathogenic Escherichia coli; lipid raft; TER; tight junction; tight junction protein Correspondence J. Li, Institute of General Surgery, Jinling Hospital, 305 East Zhongshan Road, Nanjing 210002, China Fax: +86 25 84803956 Tel: +86 25 80860064 E-mail: liqiurong@yahoo.com (Received 22 July 2008, revised 28 September 2008, accepted 6 October 2008) doi:10.1111/j.1742-4658.2008.06731.x Enteropathogenic Escherichia coli (EPEC) has been shown to disrupt the barrier function of host intestinal epithelial tissues through entering tight junctions. However, the mechanism by which this occurs remains poorly understood. In this study, we determined that EPEC invades host cells through tight junctions as it initiates infection. Immunofluorescence micro- scopy revealed redistribution of the tight-junction proteins occludin and ZO-1 from an intercellular to a cytoplasmic location after EPEC invasion. Flotillin-1 was recruited to sites of EPEC entry. EPEC entered host cells through tight-junction membrane microdomains. Tight-junction ultrastruc- ture was disrupted following EPEC infection, accompanied by loss of barrier function. EPEC infection caused a time-dependent decrease in trans-epithelial electrical resistance. Subcellular fractionation using discon- tinuous sucrose density gradients demonstrated a decline in raft-associated occludin following exposure to EPEC. These results indicate the important role of host membrane tight-junction microdomains in EPEC invasion. Abbreviations EPEC, enteropathogenic Escherichia coli;M-b-CD, methyl-b-cyclodextrin; TER, transepithelial electrical resistance; TJ, tight junction. 6022 FEBS Journal 275 (2008) 6022–6032 ª 2008 The Authors Journal compilation ª 2008 FEBS and reoviruses are known to initiate infection via TJs [10]. Microorganisms utilize lipid rafts in order to exert their effects on host cells [11–13]. However, the role of membrane rafts in the internalization of bacteria into host cells during invasion has not yet been identified clearly. The route followed by EPEC through the TJ remains poorly understood. Membrane microdomains of the TJ may be used by EPEC to penetrate the epithelial barrier. In this study, the specific involvement of TJ mem- brane microdomains in EPEC invasion into host cells was determined. The results demonstrate that TJ mem- brane microdomains are required for bacterial inva- sion, and that the distribution of TJ proteins in TJ membrane microdomains is altered in response to EPEC infection. EPEC invasion has a significant effect on localization of TJ proteins in TJ membrane microd- omains. Infection of intestinal epithelial cells with EPEC disrupts TJ structure and barrier function by altering the distribution of TJ proteins in TJ mem- brane microdomains. Results EPEC co-localizes with flotillin-1 at the entry site In order to investigate the role of lipid rafts in EPEC invasion of epithelial cells, we used confocal fluores- cence microscopy to observe the entry of the bacteria into epithelial cells, and confirmed that penetration of EPEC through TJ membrane microdomains was related to infection. We examined the role of a known lipid raft protein, flotillin-1, in EPEC invasion using a specific probe for flotillin-1. As shown in Fig. 1, we found that flotillin-1 is intimately associated with invading bacteria at the point of attachment in EPEC- infected cells. Accumulation of flotillin-1 was observed at the entry foci and around intracellular EPEC. The bacteria co-localized with flotillin-1 in the plasma membrane and also in the cytoplasm (Fig. 1), where a noticeable shift in cellular flotillin-1 to the location of intracellular bacteria was observed. Flotillin-1 accumu- lated at the sites of EPEC entry, and was transported Flotillin-1 EPEC Merge 20 min 40 min 60 min 80 min Fig. 1. EPEC co-localizes with flotillin-1. Cell monolayers exposed to EPEC for 20-80 min were stained with antibodies to flotillin-1 (red). EPEC (green) was visualized using a CFDA SE cell tracer kit. Flotillin-1 accumu- lated at the attachment site and was recruited around the internalized bacteria. Arrows indicate co-localization (yellow) of EPEC (green) with flotillin-1 (red). These experiments were repeated three times with three replicates in each experiment. Q. Li et al. EPEC enters host cells through tight junctions FEBS Journal 275 (2008) 6022–6032 ª 2008 The Authors Journal compilation ª 2008 FEBS 6023 from the apical cell surface to the TJ, and then to the cytoplasm. The lipid raft marker protein was recruited to the sites of entry of the bacteria, indicating that lipid rafts are involved in bacterial invasion. The asso- ciation of lipid rafts with bacteria revealed that EPEC entered host cells by a lipid raft-dependent route. These results indicated that, once bacteria have reached the TJ, the lipid raft is responsible for inter- nalization and invasion of EPEC. Thus, lipid rafts are required for EPEC penetration into the TJ and subse- quent infection. EPEC infection alters the distribution of TJ proteins We explored the impact of EPEC infection on the dis- tribution of occludin and ZO-1. Caco-2 cells were infected with EPEC for 1, 3 or 5 h, and then immuno- stained for occludin and ZO-1. A chicken-wire pattern of distribution for occludin and ZO-1 was seen in uninfected control monolayers (Fig. 2A,B). In unin- fected cells, occludin and ZO-1 were found to be primarily localized to the cell membrane of the TJ. Determination of the localization of these two proteins showed a continuous and uniform distribution pattern at the apical membrane of the cells and revealed marked changes in the distribution of TJ proteins fol- lowing EPEC infection that progressed in severity. Breaks in occludin staining were seen (Fig. 2A) as pre- viously reported [14]. EPEC infection of monolayers resulted in redistribution of occludin from the lateral membrane to the cytoplasm, with more pronounced changes at 5 h post-infection. Consistent with these results, the distribution of ZO-1 was also altered following EPEC infection (Fig. 2B). These findings suggest that occludin and ZO-1 dissociate from the membrane and are redistributed to the cytoplasm in response to EPEC infection. Immunofluorescence microscopy revealed accumulation of ZO-1 around the internalized EPEC (Fig. 3) during the entry process. EPEC invasion into host cells was accompanied by specific recruitment of TJ protein ZO-1. ZO-1 moved from the apical membrane to the TJ, and then to the cytoplasm concurrently with EPEC. These data indi- XY XZ ZO-1 Uninfected 1 h 3 h 5 h XY XZ Occludin A B Fig. 2. EPEC infection alters the distribution of TJ proteins. Uninfected cell monolayers (left column) and monolayers infected with EPEC for 1, 3 and 5 h were immunostained with antibodies to occludin or ZO-1. In uninfected monolayers, occludin and ZO-1 are primarily limited to the cell–cell interfaces, and the staining is continuous and uniform. After infection with EPEC, the distribution of occludin and ZO-1 was significantly altered and breaks in occludin and ZO-1 staining were seen. These experiments were repeated three times with three replicates in each experiment. EPEC enters host cells through tight junctions Q. Li et al. 6024 FEBS Journal 275 (2008) 6022–6032 ª 2008 The Authors Journal compilation ª 2008 FEBS cate that EPEC infection has significant effects on the distribution of TJ proteins in the cell monolayer, and that EPEC invasion into host cells occurs through TJ membrane microdomains. Cholesterol depletion is responsible for non-pathogenic E. coli entry into the cells In our study, methyl-b-cyclodextrin (M-b-CD), a cholesterol-depletion reagent that is commonly used to disrupt lipid rafts, was used to deplete membrane cholesterol. Caco-2 cells were pretreated with M-b-CD for 30 or 45 min, and incubated with non-pathogenic E. coli strain DH5a for 1 h. Bacteria were detected using a CFDA SE cell tracer kit. After treatment with M-b-CD, the typical chicken-wire pattern distribution of ZO-1 was disrupted (Fig. 4A,B, left column). More interestingly, the non-pathogenic E. coli was found to enter M-b-CD-treated cells. ZO-1 was found to bind to the internalized bacteria (Fig. 4A,B, right column). When DH5a was applied to cells in the absence of M-b-CD, the ultrastructures of TJs and the desmosome were similar to those of controls (Fig. 4D). It was also shown that ZO-1 is expressed along the lateral membranes of the cells with a continuous and uniform distribution (Fig. 4C). These results indicate that TJ morphology is not affected by DH5a. Non-pathogenic E. coli entered into the cells as a result of cholesterol depletion. Thus, the TJ membrane microdomains are responsible for bacterial entry into epithelial cells. EPEC infection disrupts TJ ultrastructure As the distribution pattern of TJ proteins was changed in EPEC-infected cells, we examined whether EPEC infection of epithelial cells disrupted TJ morphology. Transmission electron microscopy was performed on cell monolayers of uninfected and EPEC-infected cells to investigate the morphological features of EPEC invasion. Uninfected Caco-2 cells showed intact TJ and desmosome structures (Fig. 5A). Transmission electron micrographs of Caco-2 cells at various stages ZO-1 EPEC Merge 20 min 40 min 60 min 80 min Fig. 3. TJ proteins are responsible for EPEC entry into host cells. Monolayers were infected with EPEC for the indicated times and stained for ZO-1 (red) and bacteria (green) to assess bacterial entry. Yellow areas (arrows) represent spatial overlap (co-localization) of the red-stained ZO-1 with the green bacterial signal. Nuclei (blue) were visualized using 4¢,6-diamidino-2-pheny- lindole. These experiments were repeated three times with three replicates in each experiment. Q. Li et al. EPEC enters host cells through tight junctions FEBS Journal 275 (2008) 6022–6032 ª 2008 The Authors Journal compilation ª 2008 FEBS 6025 MergeZO-1 DH5a 30 min 45 min A B C 200n D Fig. 4. TJ membrane microdomains are required for non-pathogenic E. coli delivery into Caco-2 cells. Caco-2 monolayers pre- treated with 10 m M methyl-b-cyclodextrin (M-b-CD) were exposed to the non-invasive E. coli strain DH5a. Disruption of continuity in ZO-1 staining was observed (left column). Arrows indicate areas of co-localization (yel- low) of bacteria (green) with ZO-1 (red). DAPI was used to detect nuclear DNA (blue). These experiments were repeated three times with three replicates in each experiment. CBA 200 nm 200 nm 200 nm F E 200 nm D G 200 nm 200 nm 200 nm Fig. 5. Transmission micrographs of the tight junction in cells infected with EPEC. (A) TJs and desmosomes are intact in uninfected cells. (B) EPEC has not attached intimately and effaces microvilli. (C) The microvilli are lost at the site of attachment. (D,E) EPEC are attached inti- mately to the host cell membrane and TJ membrane fusions (‘kisses’) are partly lost. (F) The bacterium enters the host cell through the tight junction. Arrows indicate the location of tight junctions. Arrowheads show desmosomes. Scale bar = 200 nm. EPEC enters host cells through tight junctions Q. Li et al. 6026 FEBS Journal 275 (2008) 6022–6032 ª 2008 The Authors Journal compilation ª 2008 FEBS of EPEC infection show alterations of TJ ultrastruc- ture (Fig. 5B–G) characterized by the loss of microvilli at sites of attachment, intimate attachment of bacteria to the host cell membrane, and formation of surface structures embracing the bacteria. In particular, TJ membrane fusion ‘kisses’ were partly lost following EPEC entry (Fig. 5D–F). EPEC infection reduces transepithelial electrical resistance These morphological changes were correlated with dys- function of the TJ barrier as determined by measuring transepithelial electrical resistance (TER) (Fig. 6A). Cell monolayers grown on Transwell filters were exposed to EPEC, and TER was measured at the indicated time points. EPEC infection significantly decreased TER in a time-dependent manner, with mean reductions in TER of 42.9 ± 12.2 and 61.1 ± 9.6% at 2 and 6 h post-infection, respectively. The influence of cholesterol depletion on the TER of cell monolayers was also examined (Fig. 6B). The low- est concentration of M-b-CD (2 mm) caused only a moderate decrease in TER of 21.1 ± 6% at 5 h. How- ever, the TER was reduced by 54.6 ± 7.9 and 60.6 ± 3.1% after treatment with 5 and 10 mm M-b-CD, respectively, for 5 h. EPEC infection causes redistribution of TJ proteins in TJ membrane microdomains We next examined whether EPEC affected the molecu- lar organization of the TJ proteins, and further investi- gated possible molecular aspects of TJ membrane microdomains with respect to the entry of EPEC into host cells. TJ membrane microdomains in infected cell monolayers were fractionated by discontinuous sucrose density gradients. Following sucrose density gradient centrifugation, the majority of the flotillin-1 (approxi- mately 75% by densitometry) was found in Triton X-100-insoluble fractions (fraction 4) of unin- fected cells (Fig. 7A), which confirmed localization of TJ membrane microdomains in the sucrose gradients. Redistribution of flotillin-1 was detected in EPEC- infected cells (Fig. 7A). Flotillin-1 shifted from Triton X-100-insoluble fractions (fraction 4) to Tri- ton X-100-soluble fractions (fractions 6–9) following EPEC infection. Densitometric analysis of flotillin-1 bands indicated that fractions 6–9 contain 61.7% and 100% of total flotillin-1 after 1 and 5 h infection, respectively (Fig. 7B). In uninfected cells, occludin was detected both in Triton X-100-insoluble fractions (fractions 3–5) and Triton X-100-soluble fractions (fractions 6–9) (Fig. 7C). There was a change in the distribution of occludin in TJ membrane microdomains after infec- tion with EPEC compared to uninfected cells. The amount of occludin in fractions 3-5 from cells infected with EPEC decreased by 22.1% after 1 h of infection compared with uninfected cells (Fig. 7D). After 5 h infection, all the occludin was found in Triton X-100-soluble fractions (Fig. 7C). EPEC inva- sion induced displacement of occludin from deter- gent-insoluble fractions into detergent-soluble fractions, and thus may be associated directly with changes in the structure, function and morphology of TJs induced by EPEC. However, claudin-1 and -4 were not detected in Triton X-100-insoluble fractions but were present in Triton X-100-soluble fractions. The distribution of claudin-1 and -4 was almost 0 20 40 60 80 100 120 140 01234567 Control EPEC Time (h) A *** *** *** *** 0 20 40 60 80 100 120 140 0123456 Control M-β-CD 10 m M M-β-CD 5 m M M-β-CD 2 m M TER (%) TER (%) Time (h) B *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** Fig. 6. EPEC invasion decreases TER in epithelial cell monolayers. (A) Caco-2 monolayers were infected with EPEC, and TER was measured at hourly time intervals. (B) Influence of increasing con- centrations of M-b-CD on monolayer TER. Monolayers were treated with increasing concentrations of M-b-CD (M-b-CD was added to both the apical and basolateral chambers of the Transwells). Aster- isks indicate that the TER value for monolayers infected with EPEC (A) or treated with M-b-CD (B) was significantly less than that for the control (***P < 0.001) at the same time point. Data are means ± SEM from three experiments. Q. Li et al. EPEC enters host cells through tight junctions FEBS Journal 275 (2008) 6022–6032 ª 2008 The Authors Journal compilation ª 2008 FEBS 6027 unchanged after infection with EPEC for 5 h (Fig. 7E,F). These results may indicate that claudin-1 and -4 are not involved in the process of EPEC invasion, or that the presence of bacteria did not influence the localization of these proteins in TJ membrane microdomains. Thus, redistribution of TJ Control EPEC 30 min EPEC 1 h EPEC 5 h Fraction 23456789 TX-100 insoluble TX-100 soluble A 0 20 40 60 80 100 120 3–5 6–9 Control EPEC 30 min EPEC 1 h EPEC 5 h % of density in fraction 2–9 Fraction B Control EPEC 30 min EPEC 1 h EPEC 5 h Fraction 23456789 TX-100 insoluble TX-100 soluble C 0 20 40 60 80 100 120 3–5 6–9 Control EPEC 30 min EPEC 1 h EPEC 5 h % of density in fraction 2–9 Fraction D Control EPEC 30 min EPEC 1 h EPEC 5 h Fraction 23456789 Fraction 23456789 E TX-100 insoluble TX-100 soluble F Control EPEC 1 h EPEC 5 h TX-100 insoluble TX-100 soluble EPEC 30 min Fig. 7. Redistribution of flotillin-1 and the TJ proteins of occludin and claudins in TJ membrane microdomains by EPEC infection. Mono- layers were exposed to EPEC for the indicated times and lysed. TJ membrane microdomains were isolated by sucrose gradient centrifu- gation. Equal amounts of protein from each fraction were subjected to SDS–PAGE and analyzed by immunoblotting with mAb specific for flotillin-1, occludin and claudin-1 and -4. (A) EPEC infection changes the distribution of flotillin-1 in TJ membrane microdomains. The immunoblots were analyzed quantitatively (B), and the amount of flotillin-1 in raft fractions 3–5 is shown as a percentage of the total density detected in fractions 2–9. Results are means ± SEM. (C) EPEC infection displaced occludin from Triton X-100-insoluble fractions to Triton X-100-soluble fractions. (D) Densitometric analysis of occludin distribution in fractions 2–9. (E,F) Distribution of claudin-1 and claudin-4 in EPEC-infected cells. EPEC enters host cells through tight junctions Q. Li et al. 6028 FEBS Journal 275 (2008) 6022–6032 ª 2008 The Authors Journal compilation ª 2008 FEBS proteins from TJ membrane microdomains is required for invasion of EPEC into host cells and subsequent infection. Discussion An increasing number of pathogens, including some bacteria, viruses and parasites, have been found to enter host cells through lipid rafts [15–17]. Recently, it has been suggested that lipid rafts represent a spe- cial membrane microdomain that may facilitate virus entry [18]. Some bacteria have been found to interact with lipid rafts of the host plasma membrane. The mechanisms that underlie this interaction are starting to be unraveled. As EPEC invade the gastrointestinal tract, they must cross the TJ barrier in epithelial cells. The entry of EPEC into cells is a complex process, and the underlying molecular and cellular mechanisms of EPEC-induced disruption of the epithelial barrier are not completely understood. In this study, we demonstrated the importance of TJ membrane microdomains in invasion of EPEC into host cells through epithelial TJs. We have shown that EPEC infection induces a dramatic redistribution of TJ proteins along the lateral membrane, with a progressive loss in barrier function. In addition, EPEC-induced disruption in TJ structures is associ- ated with the redistribution of TJ proteins in TJ membrane microdomains. A crucial role of TJs is to prevent commensal and pathogenic microbes from entering the hosts. The effect of EPEC infection on TJ barrier function is well documented [14,19]; however, the mechanisms and molecular changes that induce the disruption are unclear. In this study, we utilized a combination of biochemical, immunofluorescence and ultrastructural analyses to characterize the effect of EPEC on TJ structure. We found that infection of intestinal epithe- lial cells with EPEC caused dramatic disruption of TJ structure with a decrease in TER. This was accom- panied by a redistribution of TJ proteins along the lateral membrane. TJ protein complexes play a key role in maintenance of intestinal barrier integrity [1,20]. EPEC invasion led to a redistribution of TJ structural proteins in TJ membrane microdomians (Fig. 2). Our data indicate that the ultrastructural alterations of TJs most likely account for redistribu- tion of occludin and flotillin-1 in TJ membrane micro- domians in EPEC-infected cells. This is the first report demonstrating that loss of TJ barrier function following EPEC infection is correlated with displace- ment of TJ proteins. The distribution of occludin in TJ membrane microdomians were altered in EPEC- infected cells, but claudins were minimally affected (Fig. 7E,F). EPEC infection induced marked decreases in the TER with a concomitant selective loss of occludin, but not claudins, from TJ membrane micr- odomians to the cytosolic compartment. It has been suggested that loss of occludin constitutes the most significant alteration in TJ transmembrane proteins, and our results are similar to those reported previ- ously [21]. EPEC infection of intestinal epithelial cells triggers two major functional effects: inflammation and disruption of barrier function. We have previ- ously shown using an in vitro model that proinflam- matory cytokines disrupt epithelial barrier function by altering lipid composition in membrane microdomains of TJ and displacing occludin from TJ membrane microdomains to detergent-soluble fractions. The dis- tribution of claudin isoforms was unaffected by cyto- kine treatment [22]. Molecular evidence regarding the inflammatory response caused by EPEC is consistent with these results [22]. In the previous study, it was reported that EPEC applied to fibroblast cells could also cause barrier function injury characterized by formation of an actin ‘pedestal’ resulting from rear- rangement of the host cytoskeleton beneath adherent bacteria [23]. Lipid rafts are the preferred entry sites for several invasive pathogens including Salmonella, Shigella, Listeria and Chlamydia [24]. It is likely that EPEC also utilizes lipid rafts as the entry point. Our study shows clearly using confocal microscopy that intracel- lular EPEC is surrounded by vesicles enriched in the lipid raft component flotillin-1 (Fig. 1). The results indicate that lipid rafts are required for EPEC invasion. Fluorescence microscopy of epithelial cell monolayers using ZO-1-specific antibody revealed accumulation of ZO-1 around internalized EPEC after exposure to EPEC (Fig. 3), implying that TJ proteins are specifically mobilized to sites of bacterial entry. Methyl-b-cyclodextrin is commonly used to specifically disrupt the structure of lipid rafts by depleting cholesterol from cells. The change induced by M-b-CD allowed non-pathogenic bacteria to recruit of ZO-1 for penetration through epithelial monolayers into cells. This observation also indicates that TJ membrane microdomains are required for bacterial invasion. Taken together, these results indicate that EPEC utilizes TJ membrane microdomains of plasma mem- branes for penetration into host cells. Internalization of EPEC occurred in TJ membrane microdomains, and the barrier function of TJ was disrupted after exposure to EPEC. Our results suggest for the first time that TJ membrane microdomains serve as Q. Li et al. EPEC enters host cells through tight junctions FEBS Journal 275 (2008) 6022–6032 ª 2008 The Authors Journal compilation ª 2008 FEBS 6029 the portal of EPEC entry into host cells. The TJ membrane microdomains have been shown to mediate internalization of EPEC into the host cell and hence infection events. Experimental procedures Antibodies and reagents Flotillin-1 mAb was purchased from BD Transduction Lab- oratories (Lexington, KY, USA). Occludin and ZO-1 were purchased from Zymed Laboratories Inc. (San Francisco, CA, USA). Alexa Fluor 635 secondary antibody, 4¢,6-di- amidino-2-phenylindole (DAPI) nucleic acid stain and the Vybrant CFDA SE cell tracer kit were purchased from Molecular Probes (Eugene, OR, USA). Complete protease inhibitor tablets were purchased from Boehringer Mann- heim (Indianapolis, IN, USA). The ECL western blotting analysis system was purchased from Amersham (Piscata- way, NJ, USA). Cell culture Caco-2 cells (American Type Culture Collection, Rockville, MD, USA) were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated newborn calf serum and antibiotics (100 UÆmL )1 penicillin, 100 lgÆmL )1 streptomycin) at 37 °C in a humidified atmo- sphere of 5% CO 2 . Before infection, the cells were placed in antibiotic-free medium with 0.5% newborn calf serum overnight. Bacterial growth and infection of host cells The wild-type strain EPEC 2348 ⁄ 69 was a generous gift from J. Kaper and J. Michalski (Center for Vaccine Development, University of Maryland, Baltimore, MD, USA). The non- pathogenic strain of E. coli DH5a was also used in this study. Wild-type EPEC 2348 ⁄ 69 and E. coli DH5a were grown overnight in Luria–Bertani broth. On the day of experimentation, the bacterial cultures were diluted into antibiotic-free cell culture medium containing 0.5% new- born calf serum and 0.5% mannose. The bacteria were grown at 37 °C in a shaking incubator overnight. The bacterial suspension was then centrifuged at 1500 g for 10 min at room temperature and resuspended in culture medium. Bacteria were added to the apical surface of the cells on Transwell filters (CoStar, Cambridge, MA, USA) to a final concentration of approximately 5 · 10 7 colony- forming units per mL, corresponding to a multiplicity of infection of 25 (25 bacteria per cell). Monolayers and bacteria were then co-incubated in antibiotic-free medium for specified times as indicated. Immunofluorescence microscopy Caco-2 cells were infected as described above. The bacteria were preincubated with the CFDA SE cell tracer kit for 1 h following the manufacturer’s instructions. For M-b-CD treatment, cell monolayers were washed with NaCl ⁄ P i and incubated with 10 mm M-b-CD for 1 h. The monolayers were then co-incubated with non-pathogenic E. coli DH5a. Control and infected monolayers of Caco-2 cells were washed with ice-cold NaCl ⁄ P i to remove non-adherent bac- teria, fixed in 3.7% paraformaldehyde in NaCl ⁄ P i , pH 7.4, for 15 min at room temperature, and permeabilized in 0.2% Triton X-100 for 10 min. Cells were washed three times with cold NaCl ⁄ P i and blocked in 5% goat serum for 10 min. Monolayers were then labeled with anti-occludin (1 : 100) or anti-ZO-1 (1 : 100) serum overnight, and then stained with Alexa Fluor 635-labeled goat anti-mouse sec- ondary IgG (1 : 100) for 1 h. After rinsing, the specimens were examined using an LSM510 laser scanning confocal microscope (Zeiss, Jena, Germany). Transmission electron microscopy The EPEC-infected cell monolayers were washed, fixed with 2.5% glutaraldehyde and then post-fixed with 1% OsO 4 , embedded in Epon 812 (Fluka AG, Buchs, Switzerland) and thin-sectioned. Sections were stained with 2% uranyl acetate and 0.2% lead citrate and viewed with an electron microscope (H-600, Hitachi, Tokyo, Japan). Transepithelial electrical resistance measurement Confluent monolayers grown on 0.33 cm 2 Transwell filters were used for TER assessment. TER was measured using an EVOM epithelial volt-ohm meter (World Precision Instruments, Stevenage, UK) as previously described [25]. Results are expressed as a percentage of initial resistance. Isolation of TJ membrane microdomains and Western blot analysis After bacterial infection, cells were washed with NaCl ⁄ P i and harvested by scraping into ice-cold lysis buffer (50 mm Tris, 25 mm KCl, 5 mm MgCl 2 Æ6H 2 O, 2 mm EDTA, 40 mm NaF, 4mm Na 3 VO 4 , pH 7.4, containing 1% Triton X-100 and a protease inhibitor mixture). TJ membrane microdomain frac- tions were isolated as previously described [4]. Protein con- centrations were determined by the Bradford method. Aliquots of equal protein content were isolated by SDS–PAGE and transferred to poly(vinylidene difluoride) membranes as previously described [22]. Western blots were quantified by densitometric analysis using quantity one 1d analysis software (Bio-Rad, Hercules, CA, USA). EPEC enters host cells through tight junctions Q. Li et al. 6030 FEBS Journal 275 (2008) 6022–6032 ª 2008 The Authors Journal compilation ª 2008 FEBS Statistical analysis Data are expressed as means ± SEM. The significance of differences was determined using a paired Student’s t test. P values < 0.05 were considered to be statistically significant. Acknowledgements This work was supported by grants from the National Basic Research Program (973 Program) in China (numbers 2007CB513005 and 2009CB522405) and the Key Project of the National Natural Science Founda- tion in China (30830098) to J. L., and from the National Natural Science Foundation in China (30672061) and the Key Project of Nanjing Military Command (06Z40) and the Military Scientific Research Fund (0603AM117) to Q. L. We would like to thank the Deutscher Akademischer Austauschdienst Researcher Fellowship (Bioscience Special Program, Germany) for support of Q. L., and James Kaper and Jane Michalski (Center for Vaccine Development, University of Maryland, Baltimore) for the generous gift of EPEC strain 2348 ⁄ 69. References 1 Schneeberger EE & Lynch RD (2004) The tight junc- tion: a multifunctional complex. Am J Physiol 286, C1213–C1228. 2 Balkovetz DF & Katz J (2003) Bacterial invasion by a paracellular route: divide and conquer. Microbes Infect 5, 613–619. 3 Muza-Moons MM, Schneeberger EE & Hecht GA (2004) Enteropathogenic Escherichia coli infection leads to appearance of aberrant tight junctions strands in the lateral membrane of intestinal epithelial cells. Cell Microbiol 6, 783–793. 4 Nusrat A, Parkos CA, Verkade P, Foley CS, Liang TW, Innis-Whitehouse W, Eastburn KK & Madara JL (2000) Tight junctions are membrane microdomains. J Cell Sci 113, 1771–1781. 5 Knodler L, Vallance B, Hensel M, Ja ¨ ckel D, Finlay BB & Steele-Mortimer O (2003) Salmonella type III effec- tors PipB and PipB2 are targeted to detergent-resistant microdomains on internal host cell membranes. Mol Microbiol 49, 685–704. 6 Lafont F, Tran Van Nhieu G, Hanada K, Sansonetti P & van der Goot F (2002) Initial steps of Shigella infection depend on the cholesterol ⁄ sphingolipid raft-mediated CD44–IpaB interaction. EMBO J 21, 4449–4457. 7 Seveau S, Bierne H, Giroux S, Prevost M & Cossart P (2004) Role of lipid rafts in E-cadherin- and HGF- R ⁄ Met-mediated entry of Listeria monocytogenes into host cells. J Cell Biol 166, 743–753. 8 Jutras I, Abrami L & Dautry-Varsat A (2003) Entry of the lymphogranuloma venereum strain of Chlamydia trachomatis into host cells involves cholesterol-rich membrane domains. Infect Immun 71, 260–266. 9 Wooldridge KG, Williams PH & Ketley JM (1996) Host signal transduction and endocytosis of Campylo- bacter jejuni. Microb Pathog 21, 299–305. 10 Coyne CB & Bergelson JM (2006) Virus-induced Abl and Fyn kinase signals permit coxsackievirus entry through epithelial tight junctions. Cell 124, 119–131. 11 Duncan MJ, Shin JS & Abraham SN (2002) Microbial entry through caveolae: variations on a theme. Cell Microbiol 4, 783–791. 12 Lafont F & van der Goot FG (2005) Bacterial invasion via lipid rafts. Cell Microbiol 7, 613–620. 13 Simons K & Ehehalt R (2002) Cholesterol, lipid rafts and disease. J Clin Invest 110, 597–603. 14 Simonovic I, Rosenberg J, Koutsouris A & Hecht G (2000) Enteropathogenic Escherichia coli dephosphory- lates and dissociates occludin from intestinal epithelial tight junctions. Cell Microbiol 2, 305–315. 15 Haldar K, Mohandas N, Samuel BU, Harrison T, Hil- ler NL, Akompong T & Cheresh P (2002) Protein and lipid trafficking induced in erythrocytes infected by malaria parasites. Cell Microbiol 4, 383–395. 16 Pelkmans L & Helenius A (2003) Insider information: what viruses tell us about endocytosis. Curr Opin Cell Biol 15, 414–422. 17 Lafont F, Abrami L & van der Goot FG (2004) Bacterial subversion of lipid rafts. Curr Opin Microbiol 7, 4–10. 18 Takeda M, Leser GP, Russell CJ & Lamb RA (2003) Influenza virus hemagglutinin concentrates in lipid raft microdomains for efficient viral fusion. Proc Natl Acad Sci USA 100, 14610–14617. 19 Spitz J, Yuhan R, Koutsouris A, Blatt C, Alverdy J & Hecht G (1995) Enteropathogenic Escherichia coli adherence to intestinal epithelial monolayers diminishes barrier function. Am J Physiol 268, G374–G379. 20 Mitic LL, Van Itallie CM & Anderson JM (2000) Molecular physiology and pathophysiology of tight junctions I. Tight junction structure and function: les- sons from mutant animals and proteins. Am J Physiol 279, G250–G254. 21 McNamara BP, Koutsouris A, O’Connell CB, Nou- gayre ´ de JP, Donnenberg MS & Hecht G (2001) Trans- located EspF protein from enteropathogenic Escherichia coli disrupts host intestinal barrier function. J Clin Invest 107, 621–629. 22 Li QR, Zhang Q, Wang M, Zhao SM, Ma J, Luo N, Li N, Li YS, Xu GW & Li JS (2008) Interferon-c and tumor necrosis factor-a disrupt epithelial barrier func- tion by altering lipid composition in membrane micro- domains of tight junction. Clin Immunol 126, 67–80. Q. Li et al. EPEC enters host cells through tight junctions FEBS Journal 275 (2008) 6022–6032 ª 2008 The Authors Journal compilation ª 2008 FEBS 6031 [...]...EPEC enters host cells through tight junctions Q Li et al 23 Rohde M (2007) Bacterial pathogenesis: insights into a new world discovered by high-resolution field emission scanning electron microscopy (FESEM) In Research Report 2006 ⁄ 2007: Special Features pp 46–53... Manes S, del Real G & Martinez AC (2003) Pathogens: raft hijackers Nat Rev Immunol 3, 557–568 25 Li QR, Zhang Q, Wang M, Zhao SM, Xu GW & Li JS (2008) n-3 polyunsaturated fatty acids prevent disruption of epithelial barrier function induced by proinflammatory cytokines Mol Immunol 45, 1356–1365 FEBS Journal 275 (2008) 6022–6032 ª 2008 The Authors Journal compilation ª 2008 FEBS . Invasion of enteropathogenic Escherichia coli into host cells through epithelial tight junctions Qiurong Li 1,2 , Qiang. October 2008) doi:10.1111/j.1742-4658.2008.06731.x Enteropathogenic Escherichia coli (EPEC) has been shown to disrupt the barrier function of host intestinal epithelial tissues through entering tight junctions.