Invasion of Epithelial Cells by Locus of Enterocyte Effacement-Negative Enterohemorrhagic Escherichia coli
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感染与免疫杂志 2005年第5期
Australian Bacterial Pathogenesis Program, Department of Microbiology, Monash University, Victoria 3800
Australian Bacterial Pathogenesis Program, Department of Microbiology and Immunology, University of Melbourne, Victoria 3010
Microbiological Research Unit, Murdoch Childrens Research Institute and Royal Childrens Hospital, Parkville Victoria 3052, Australia
ABSTRACT
The majority of enterohemorrhagic Escherichia coli (EHEC) strains associated with severe disease carry the locus of enterocyte effacement (LEE) pathogenicity island, which encodes the ability to induce attaching and effacing lesions on the host intestinal mucosa. While LEE is essential for colonization of the host in these pathogens, strains of EHEC that do not carry LEE are regularly isolated from patients with severe disease, although little is known about the way these organisms interact with the host epithelium. In this study, we compared the adherence properties of clinical isolates of LEE-negative EHEC with those of LEE-positive EHEC O157:H7. Transmission electron microscopy revealed that LEE-negative EHEC O113:H21 was internalized by Chinese hamster ovary (CHO-K1) epithelial cells and that intracellular bacteria were located within a membrane-bound vacuole. In contrast, EHEC O157:H7 remained extracellular and intimately attached to the epithelial cell surface. Quantitative gentamicin protection assays confirmed that EHEC O113:H21 was invasive and also showed that several other serogroups of LEE-negative EHEC were internalized by CHO-K1 cells. Invasion by EHEC O113:H21 was significantly reduced in the presence of the cytoskeletal inhibitors cytochalasin D and colchicine and the pan-Rho GTPase inhibitor compactin, whereas the tyrosine kinase inhibitor genistein had no significant impact on bacterial invasion. In addition, we found that EHEC O113:H21 was invasive for the human colonic cell lines HCT-8 and Caco-2. Overall these studies suggest that isolates of LEE-negative EHEC may employ a mechanism of host cell invasion to colonize the intestinal mucosa.
INTRODUCTION
Infection with enterohemorrhagic Escherichia coli (EHEC) can result in a variety of clinical conditions, ranging in severity from mild diarrhea to bloody diarrhea and the hemolytic uremic syndrome (40). Strains of EHEC that carry the locus of enterocyte effacement (LEE) are associated with progression to severe disease and exhibit a distinctive mechanism of colonization that is characterized by intimate bacterial adherence to the host intestinal mucosa and the formation of attaching and effacing (A/E) lesions (20). The ultrastructural changes characteristic of A/E lesions result from cytoskeletal rearrangements in the host cell, in particular the recruitment of filamentous actin beneath adherent bacteria (28). This ability to manipulate the host cytoskeleton and induce intimate bacterial attachment is encoded by the LEE pathogenicity island of A/E pathogens such as EHEC and the closely related pathogen enteropathogenic E. coli (EPEC) (36). In these pathogens LEE is essential for colonization of the host and virulence (32, 37, 48, 51).
Throughout infection A/E pathogens remain largely restricted to the mucosal surface, and they are generally regarded as extracellular pathogens. In contrast, other enteric pathogens, such as Yersinia, Shigella, and Salmonella spp., colonize the host by invading the intestinal mucosa. Salmonella and Shigella spp. exploit the process of macropinocytosis to effect host cell entry and rely on a functional host cell cytoskeleton as well as activation of members of the Rho family of GTPases to stimulate their uptake (1, 9). The activation of Cdc42 and Rac induces host cell cytoskeletal rearrangements and membrane ruffling that results in engulfment and internalization of the bacteria (21, 38). This activation is mediated by homologous bacterial secreted effector proteins that are translocated into the host cell by type III secretion systems (21). Inhibitors of Rho GTPase activity and cytoskeletal inhibitors of actin filament formation block Salmonella- and Shigella-induced invasion (17, 55). In contrast, invasin-mediated uptake of the enteropathogenic Yersinia spp. by epithelial cells depends on high-affinity interaction between the bacterial outer membrane protein invasin and host cell 1-integrins (25). Clustering of the host cell receptor and the subsequent activation of focal adhesion kinase by tyrosine phosphorylation lead to internalization of the adherent bacteria in a process similar to receptor-mediated endocytosis (2, 53). Like that by Salmonella and Shigella, invasion by Yersinia is blocked by inhibitors of actin filament formation (57). However, whereas the tyrosine kinase inhibitor genistein blocks invasion by Yersinia, it has no effect on the internalization of Salmonella by epithelial cells (47, 52). These organisms therefore exploit distinct host cell processes to stimulate their uptake.
Although the majority of EHEC strains isolated from patients are A/E pathogens, including the O157:H7 serotype, some isolates of EHEC do not carry LEE and are not A/E pathogens (44). These strains have been termed LEE-negative or eae-negative EHEC or shiga toxigenic E. coli, and they have been regularly associated with sporadic cases and small outbreaks of hemorrhagic colitis and the hemolytic uremic syndrome worldwide (6, 16, 23, 27, 43). An early study investigating the adherence mechanisms of LEE-negative EHEC O113:H21 showed that in contrast to EHEC O157:H7, EHEC O113:H21 adhered to HEp-2 human epithelial cells without actin accumulation or A/E lesion formation (15). Transmission electron microscopy of infected HEp-2 cells revealed that the bacteria attached to the epithelial cell surface in abundance and that some bacteria were located within intracellular vacuoles. More recent studies have shown that EHEC O113:H21 also adheres to Henle 407 and Chinese hamster ovary (CHO-K1) cells (14, 42) and that LEE-negative EHEC O91:H21 adheres to human intestinal T84 cells and has the capacity to colonize the intestinal tracts of mice and cattle (13, 31, 49). Two putative adhesins have been identified that promote attachment to host cells in tissue culture. These include Saa, an autoagglutinating adhesin similar to YadA from Yersinia spp., which is encoded by the large hemolysin plasmid pO113, and LPFO113, a putative fimbrial adhesin encoded by a chromosomal gene cluster (14, 42). The aim of this work was to continue characterization of the LEE-negative EHEC adherence phenotype and compare the interactions of EHEC O113:H21 and EHEC O157:H7 with CHO-K1 cells and the human colonic cell lines HCT-8 and Caco-2.
MATERIALS AND METHODS
Bacterial strains and growth conditions. The bacterial strains used in this study are listed in Table 1. E. coli strains were grown in Luria-Bertani (LB) broth and incubated aerobically with shaking at 37°C overnight. EHEC strains EH5, EH42, and EH71 were tested for the production of EHEC hemolysin by growth on agar containing washed sheep erythrocytes, and zones of lysis were assessed at 8 h and after overnight incubation at 37°C. Stx production and stx typing were performed as described previously (4).
Cell lines and cell culture. CHO-K1 cells were maintained in alpha modified Eagle's medium (Invitrogen/Gibco, Carlsbad, CA) supplemented with 10% fetal calf serum (FCS) and 10 mM HEPES. Human colonic HCT-8 cells were maintained in RPMI 1640 (Invitrogen/Gibco) supplemented with 10% FCS, 1 mM sodium pyruvate, 2 mM L-glutamine, and 10 mM HEPES. The Caco-2 cell line was maintained as undifferentiated cells in Dulbecco's modified Eagle's medium (ThermoTrace, Noble Park, Victoria, Australia) containing 20% FCS, 9 mM sodium bicarbonate, 2 mM L-glutamine, 20 mM HEPES, 40 μg/ml gentamicin, and 2 μg/ml amphotericin B. All cell lines were incubated at 37°C in the presence of 5% CO2.
Quantitative invasion assays and eukaryotic cell inhibitors. Quantitative assessment of bacterial invasion of CHO-K1, HCT-8, and Caco-2 cells was done as described previously (46). Briefly, washed semiconfluent cell monolayers were infected in the presence of 0.5% mannose with around 107 CFU of each bacterial test strain for 3 h, after which some cell monolayers were washed five times with phosphate-buffered saline (PBS) and lysed in 0.1% digitonin. Following cell lysis, bacteria were resuspended in LB broth and quantified by plating serial dilutions to give the total number of cell-associated bacteria. To obtain the number of intracellular bacteria, a second set of infected wells was washed five times and incubated with 100 μg/ml gentamicin for 60 min. Following this incubation period, cells were washed five times with PBS, lysed with 0.1% digitonin, and resuspended in LB broth for quantification by plating serial dilutions. Assays were carried out in duplicate, and the results from at least three independent experiments were expressed as the percentage of the total cell-associated bacteria that were intracellular (mean ± standard deviation). Differences in invasion were assessed for significance by using an unpaired, two-tailed t test.
To assess the effect of eukaryotic cell inhibitors on bacterial invasion, cell monolayers were incubated with cytochalasin-D (1 μM), colchicine (1.25 μM), compactin (25 μM), or genistein (50 μM) (Sigma-Aldrich, St. Louis, MO) prior to bacterial inoculation as described previously (8, 33). Cytochalasin-D and colchicine were added to cells 30 min prior to inoculation, genistein 15 min prior to inoculation, and compactin 18 h prior to inoculation. All of the inhibitors were present throughout the 3-h infection period.
Electron microscopy. CHO-K1 cells were infected with EHEC O113:H21 strain EH41 or EHEC O157:H7 strain EDL933 for 3 h and fixed in 2.5% glutaraldehyde, followed by postfixation in 2.5% osmium tetroxide for 1 h. Following dehydration in a graded acetone series, the cell pellet was embedded in Epon-Araldite epoxy resin. Thin (0.5-μm) sections were stained with 10% uranyl acetate and 2.5% lead citrate before viewing under a Phillips CM12 electron microscope at 60 kV.
Antibodies and immunofluorescence microscopy. To visualize EHEC O113:H21 for immunofluorescence studies, rabbit polyclonal antibodies were raised to surface components of EHEC O113:H21. Briefly, supernatant extracts were collected by 10% trichloroacetic acid precipitation from EHEC O113:H21 EH41 grown overnight in Dulbecco's modified Eagle's medium; 100 μg of the precipitate was resuspended in PBS and Freund's complete adjuvant and used to immunize a rabbit by intramuscular inoculation. Primary immunization was followed by three booster injections at 3-week intervals using Freund's incomplete adjuvant, and the resulting antiserum was absorbed three times with whole heat-killed E. coli DH5.
For immunofluorescence, approximately 105 CHO-K1 cells or HCT-8 cells were grown on 12-mm-diameter coverslips in 24-well plates (Sarstedt, Nümbrecht, Germany). Wells were inoculated with around 5 x 107 CFU of each test strain and incubated for 5, 15, or 30 min at 37°C in 5% CO2. Following the incubation period, cells were washed five times and fixed with 10% formalin for 20 min. To visualize extracellular bacteria, coverslips were incubated at room temperature for 60 min with anti-O113:H21 diluted 1:100 in PBS containing 0.2% bovine serum albumin (PBS-BSA). Coverslips were washed three times and incubated for 60 min with a second anti-rabbit antibody conjugated to tetramethylrhodamine isothiocyanate (TRITC) (Sigma) diluted 1:200 in PBS-BSA. Following this initial staining, cells were permeabilized with 0.1% Triton X-100 for 4 min and washed three times with PBS. To stain intracellular bacteria, anti-O113:H21 antibodies were diluted 1:100 in PBS-BSA and incubated with coverslips for 60 min as before. Coverslips were then washed three times and incubated with a second anti-rabbit antibody conjugated to fluorescein-6-isothiocyanate (FITC) (Sigma) diluted 1:200 in PBS-BSA. To visualize the cell cytoskeleton, permeabilized cells were incubated with phalloidin-TRITC (0.05 μg/ml) (Sigma). All preparations were examined by epifluorescence microscopy using an Olympus BX51 microscope with a 100x oil immersion objective (Olympus, Tokyo, Japan). Digital images were acquired using an Olympus DP-70 digital camera and merged using DP manager software version 1.1.1.71.
Construction of an escF deletion mutant of EDL933. To construct an escF mutant of EDL933, the deleted escF region carrying a kanamycin resistance marker was amplified by PCR from EPEC escF ICC171 (56) by using the primers 5'-CACTGACTCGTTTGCTCG-3' and 5'-CTAATCTTGGCTAACTCTC-3' under the following conditions: 2 min at 94°C; 30 cycles of 44 s at 94°C, 40 s at 46°C, and 1 min at 72°C; and an extension period of 5 min at 72°C. The resulting PCR product was introduced into EDL933 harboring pKD46 and recombined into the EDL933 genome as described previously (12).
Detection of known invasion genes by PCR and low-stringency hybridization. Oligonucleotide primers were designed to amplify inv from Yersinia enterocolitica W22703 (5'-TGCGTACCTTCCAACAAAG-3' and 5'-GGCGTACTATCAATATTAGTC-3', covering nucleotides 439 to 1441 of the native gene), sipC from Salmonella enterica serovar Typhimurium LT2 (5'-GAGTCCTACACTGAGCGC-3' and 5'-TTGCCTGCGATAGCAGCG-3'), and ipaC from enteroinvasive E. coli (EIEC) 6/84 (5'-CAGCAGATTGCAGCGCATAT-3' and 5'-CAAGAGCAGATGCATAACGC-3') by PCR. PCR amplification was performed on 500 ng of template DNA with approximately 1 μg of each primer PCR per 100-μl reaction mixture. Amplification was performed under the following conditions: 2 min at 94°C; 30 cycles of 44 s at 94°C, 40 s at the appropriate annealing temperature (inv, 50°C; sipC, 45°C; ipaC, 50°C), and 1 min at 72°C; and an extension period of 5 min at 72°C. PCR-generated DNA products were examined by agarose gel electrophoresis. To generate digoxigenin-labeled probes, digoxigenin-dUTP was incorporated into the PCR according the manufacturer's instructions (Roche, Indianapolis, IN). Southern hybridizations were performed at low stringency (55 to 60°C) according to the manufacturer's protocol (Roche). Genomic DNA for hybridizations and PCR was isolated using the DNeasy tissue kit (Qiagen, Hilden, Germany) according the manufacturer's instructions.
RESULTS
Interaction of EHEC O113:H21 and EHEC O157:H7 with CHO-K1 cells. Previously we reported that EHEC O113:H21 strain EH41 adheres to CHO-K1 epithelial cells (14). In this study we examined this interaction more closely by comparing the adherence properties of LEE-negative EHEC with those of LEE-positive EHEC O157:H7. CHO-K1 cells were incubated with EHEC O113:H21 strain EH41 and EHEC O157:H7 strain EDL933 for 3 h and examined by transmission electron microscopy. Unexpectedly, this showed that EHEC O113:H21 was internalized and that the bacteria were frequently located within a membrane-bound vacuole (Fig. 1A and inset). Although extracellular bacteria were also seen in association with EH41-infected cells, these were located with cells that had undergone necrosis (Fig. 1C). In contrast, EHEC O157:H7 strain EDL933 remained extracellular, intimately attached to the epithelial cell surface (Fig. 1B), and extracellular bacteria were not associated with necrotic cells (Fig. 1D).
Ability of EHEC O113:H21 and other serotypes of LEE-negative EHEC to invade CHO-K1 cells. To determine if other clinical isolates of EHEC O113:H21 and other serotypes of LEE-negative EHEC were capable of invading epithelial cells, we performed quantitative invasion assays on CHO-K1 cells infected with various strains of EHEC (46). The invasive capacities of various strains and serotypes of LEE-negative EHEC were compared with those of two isolates of EIEC (which exhibit a Shigella-like mechanism of invasion), two isolates of EHEC O157:H7, and one isolate of EPEC (strain E2348/69, which has previously been reported to invade epithelial cells [19, 26]). Noninvasive E. coli HB101 and E. coli DH5 served as negative controls. All strains of LEE-negative EHEC expressed EHEC hemolysin and Stx-2, as determined previously (4, 16) or in this study. The quantitative invasion assays showed that isolates of LEE-negative EHEC were significantly more invasive for CHO-K1 cells than EHEC O157:H7, EPEC E2348/69, and E. coli HB101 and DH5 (Fig. 2). These data confirmed that EHEC O113:H21 was able to invade epithelial cells and showed that the levels of CHO-K1 cell invasion by all LEE-negative EHEC strains irrespective of serotype were comparable to those observed for EIEC strains (Fig. 2). In addition, we tested the ability of a pO113-cured derivative of EHEC O113:H21, strain EH41c, to invade epithelial cells. Plasmid-borne invasins have been well characterized for other pathogens, such as Shigella, and pO113 encodes several putative virulence determinants, including Saa, which shares similarity with the plasmid-encoded adhesin/invasin YadA from Y. enterocolitica. There was no difference, however, in the levels of invasion observed for EH41 and EH41c, indicating that pO113-encoded factors were not required to induce EH41 uptake by CHO-K1 cells, although this does not necessarily discount a role for the large hemolysin plasmid in other LEE-negative invasive strains. In addition, to account for the possibility that EH41 and EDL933 share invasion genes but that the LEE pathogenicity island contributes to an antiphagocytic effect which would result in decreased invasion, we tested the ability of an escF deletion mutant of EHEC O157:H7 EDL933 to invade CHO-K1 cells. However, there was no difference in invasion levels between wild-type EDL933 and the escF mutant strain (data not shown), indicating that LEE does not interfere with EHEC O157:H7 uptake.
Kinetics of EHEC O113:H21 invasion of CHO-K1 cells. The quantitative gentamicin protection assay used in this study indicated the number of intracellular bacteria at 3 h postinoculation. To examine the early stages of epithelial cell invasion by EHEC O113:H21, we used an immunofluorescence assay to differentiate between intracellular and extracellular bacteria at 5, 15, and 30 min postinfection. A similar approach has been used previously to examine invasion by Salmonella (50). Following incubation with bacteria, CHO-K1 cells were fixed and incubated with anti-O113:H21 antibodies and secondary antibodies conjugated to TRITC or FITC. This procedure labeled intracellular bacteria with FITC, while extracellular bacteria were labeled with both TRITC and FITC. Therefore, intracellular bacteria fluoresced green, while extracellular bacteria fluoresced yellow/orange due to colocalization of TRITC and FITC. In addition the cell cytoskeleton was stained with phalloidin-TRITC. The results showed that 5 min after inoculation, some bacteria that were associated with the host cell were located intracellularly, although the majority appeared to be surface located (Fig. 3, upper left panel). Extracellular bacteria were also visible by phase-contrast microscopy (data not shown). At 15 min and 30 min the majority of bacteria fluoresced green, indicating that invasion was largely complete (Fig. 3, upper right and lower left panels). Intracellular bacteria were not visible by phase-contrast microscopy (data not shown). Interestingly, at all time points some bacteria were observed to stain partly green and partly orange, suggesting these organisms were in the process of cell invasion (Fig. 3, lower right panel). This apparent cell entry was not associated with any obvious ruffling of the host cell membrane or focusing of the actin cytoskeleton. Again, the extracellular component of the invading bacterium was visible by phase-contrast microscopy (data not shown).
Effect of eukaryotic cell inhibitors on EHEC O113:H21 invasion of CHO-K1 cells. Many invasive pathogens exploit specific host cell pathways to induce their own uptake. To identify host cell structures and processes that were involved in epithelial cell invasion by LEE-negative EHEC, we treated CHO-K1 cells with eukaryotic cell inhibitors known to block bacterial invasion by various pathogens (8, 11, 33, 47). CHO-K1 cells were treated with cytochalasin D to disrupt actin microfilament formation or with colchicine to inhibit microtubule function and subsequently infected with EHEC O113:H21 EH41. Treatment with either of these agents blocked CHO-K1 cell invasion by strain EH41, demonstrating that a functional host cell cytoskeleton is needed for bacterial uptake (Table 2). In contrast, bacterial adherence was unaffected by the inhibitors (data not shown). To ascertain if any members of the Rho family of GTP-binding proteins played a role in internalization of EH41, CHO-K1 cells were treated with the pan-Rho GTPase inhibitor compactin. Bacterial invasion of compactin-treated CHO-K1 cells was significantly reduced compared with that of untreated cells, indicating that Rho GTPases were important for EH41-mediated internalization (Table 2). In contrast, the treatment of CHO-K1 cells with genistein had no significant impact on invasion by strain EH41 (Table 2). Again, neither inhibitor affected levels of bacterial adherence (data not shown).
Invasion of HCT-8 and Caco-2 cells by EHEC O113:H21. Although CHO-K1 cells are widely used to study bacterial interactions with epithelial cells, they do not represent a host cell type that is particularly relevant to human infection. Therefore, to ensure that invasion by EHEC O113:H21 uptake was not peculiar to CHO-K1 cells, we also infected the human colonic cell lines HCT-8 and Caco-2 with EHEC O113:H21 strain EH41. Quantitative gentamicin protection assays showed that EH41 and EIEC strain 6/84 were significantly more invasive for HCT-8 cells than EDL933 and E. coli HB101 (Table 3). As for CHO-K1 cells, the level of EH41 invasion into HCT-8 cells was not significantly different from that of EIEC 6/84. In addition, EH41 was significantly more invasive for Caco-2 cells than EHEC EDL933 and E. coli HB101 (Table 3). These results demonstrated that LEE-negative EHEC strains were invasive for epithelial cells of human intestinal origin.
The level of E. coli HB101 internalization into HCT-8 cells was surprisingly high compared with invasion levels observed in CaCo-2 or CHO-K1 cells (Fig. 1 and Table 3). We therefore repeated the quantitative invasion assays in HCT-8 and CHO-K1 cells with a strain of E. coli DH5 as a further negative control (Fig. 1 and Table 3). The results showed that while E. coli DH5 invasion in CHO-K1 cells was equivalent to that of E. coli HB101 (0.35% ± 0.26% and 0.11% ± 0.08%, respectively), in HCT-8 cells, E. coli HB101 invasion was significantly higher than that of E. coli DH5 (P = 0.034) (Table 3). This suggested that the strain of E. coli HB101 used in this study showed an unusual capacity to invade HCT-8 cells but not CaCo-2 or CHO-K1 cells. However, similar to the case for E. coli HB101, the level of E. coli DH5 internalization in HCT-8 cells was higher than that in CHO-K1 cells (1.2% ± 0.69% and 0.35% ± 0.26%, respectively), suggesting that the HCT-8 cell line also has a greater inherent capacity to take up bacteria than CHO-K1 or CaCo-2 cells.
Screening for known enterobacterial invasion determinants. As no genome sequence is available for EHEC O113:H21 or any LEE-negative EHEC strain, we tested for the presence of known invasion genes of enterobacteria by PCR and low-stringency Southern and dot blot hybridizations. The genes examined included a 5' region of inv from Y. enterocolitica, which encodes invasin, and sipC from S. enterica serovar Typhimurium and ipaC from EIEC, which encode components of their respective type III secretion systems that are essential for inducing host cell ruffling and bacterial uptake. No homologous nucleotide sequences were detected for any of these genes by PCR in the LEE-negative EHEC strains, but low-stringency hybridization revealed that three of the eight invasive strains tested, EH5, EH53, and EH71, carried sequences homologous to ipaC although not to sipC or inv (data not shown).
DISCUSSION
Attachment to the intestinal mucosa by EHEC is an essential step towards colonization of the host and the development of disease. Most clinical isolates of EHEC are A/E pathogens that carry the LEE pathogenicity island and induce well-characterized and distinctive lesions on the intestinal epithelium following intimate adherence to the enterocyte surface. However, LEE-negative EHEC strains are not A/E pathogens, and in the absence of LEE, little is known about how these strains colonize the host (44). In this study we were able to show that, in contrast to EHEC O157:H7, various serotypes of LEE-negative EHEC were invasive for CHO-K1 epithelial cells. Although EHEC O157:H7 has been reported to enter cells at a higher rate than E. coli HB101 (41), which was also true in our study, here invasion by LEE-negative EHEC strains was consistently 5- to 10-fold higher than invasion by two isolates of EHEC O157:H7. The level of invasion observed for LEE-negative EHEC strains was also significantly higher than that observed for EPEC E2348/69 and matched that of two EIEC isolates which exhibit a Shigella-like mechanism of internalization. However, unlike Shigella and EIEC, which escape from the phagosome into the cell cytoplasm, EHEC O113:H21 remained within a membrane-bound vacuole. Investigation of the kinetics of CHO-K1 cell invasion by EHEC O113:H21 showed that internalization was rapid and occurred within minutes of bacterial attachment to the cell surface. The LEE-negative EHEC invasion phenotype was also observed in the human colonic cell lines HCT-8 and Caco-2, which represent a more relevant in vitro model of infection than CHO-K1 cells. Interestingly, the level of bacterial invasion into HCT-8 cells for all strains tested was somewhat higher than the levels observed for CHO-K1 cells and Caco-2 cells, suggesting that HCT-8 cells have some inherent capacity for phagocytosis.
Many bacterial pathogens, including enteric pathogens, invade mucosal surfaces in order to colonize the host. Invasion of the intestinal mucosa is a feature of infections by Yersinia, Salmonella, and Shigella spp. Uptake of the bacteria by host cells relies on a functional host cell cytoskeleton in all cases (21). Using the cytoskeletal inhibitors cytochalasin-D and colchicine, we showed that invasion of CHO-K1 cells by EHEC O113:H21 required an intact actin cytoskeleton as well as microtubule function. Although microtubule function is not required for invasion by Salmonella, Shigella, and Yersinia (18), microtubules have been shown to be important for the uptake of other invasive pathogens (5, 8, 10, 11, 24). In addition, use of the inhibitors compactin and genistein showed that EHEC O113:H21 invasion relied upon the activity of Rho GTPases but not on that of tyrosine kinases.
Rho GTPases regulate cytoskeletal rearrangements within host cells (22). In response to contact with Salmonella spp., actin ruffling of the host cell surface, which coincides with bacterial invasion, is driven by activation of Cdc-42 and Rac. The zipper mechanism of host cell invasion by Yersinia spp., on the other hand, requires tyrosine kinase activity as well as activation of Rho GTPases, in particular Rac1 (3, 25). While the results of our experiments with inhibitors of bacterial uptake suggested that invasion by EHEC O113:H21 depended upon the same cellular processes as Salmonella-mediated uptake, we were unable to identify any membrane ruffling associated with EHEC O113:H21 entry into CHO-K1 cells by using phalloidin to stain for filamentous actin. Martinez and Hultgren have described the involvement of Rho-GTPases in the zipper mechanism of invasion of bladder epithelial cells by uropathogenic E. coli, which invades using type I pili (33), but this process also requires tyrosine kinase activity (34). Hence, the precise pathway of LEE-negative EHEC-induced entry is still unclear.
At this stage the genetic basis of invasion by LEE-negative EHEC is unknown. The inclusion of mannose in our invasion assays precluded the involvement of type I pili as described for uropathogenic E. coli and adherent-invasive E. coli, which is associated with Crohn's disease (7, 34), and although several virulence-associated factors have been identified on the large hemolysin plasmid pO113, curing of this plasmid from EHEC O113:H21 strain EH41 had no impact on CHO-K1 cell invasion, implying that the putative invasins are likely to be chromosomally encoded, at least in strain EH41. We also screened for homologues of known invasion genes, including inv, ipaC, and sipC. The presence of a putative ipaC homologue in some invasive LEE-negative EHEC strains but not others may indicate that these pathogens carry diverse invasion loci, some of which may be related to the ipa invasion locus of EIEC. Identification of the ipaC homologue in strains EH5, EH53, and EH71 would help to characterize the genetic basis of invasion in these strains, and further testing will show how prevalent this locus is among LEE-negative EHEC isolates. Nevertheless, since five of the eight LEE-negative EHEC strains tested in this study were negative for the ipaC probe, it is likely that other, as-yet-unidentified genes contribute to the invasion phenotype. Overall this study suggests that LEE-negative EHEC strains may employ a mechanism of host cell invasion to colonize the intestinal epithelium which in the pathogenesis of these infections may compensate for the absence of LEE and the inability to form A/E lesions.
ACKNOWLEDGMENTS
We are indebted to James Paton and Adrienne Paton, University of Adelaide, for the gift of the HCT-8 cell line and to Carl Kirkwood, Murdoch Children's Research Institute, for the CaCo-2 cell line. We are also grateful to Gadi Frankel, Imperial College, London, for sending us strain ICC171. We greatly appreciate the assistance of Julia Prentice in the construction of the escF deletion mutant of EDL933, and we thank Yan Tan for culturing the CaCo-2 cell line.
This work was supported by the Australian National Health and Medical Research Council.
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Australian Bacterial Pathogenesis Program, Department of Microbiology and Immunology, University of Melbourne, Victoria 3010
Microbiological Research Unit, Murdoch Childrens Research Institute and Royal Childrens Hospital, Parkville Victoria 3052, Australia
ABSTRACT
The majority of enterohemorrhagic Escherichia coli (EHEC) strains associated with severe disease carry the locus of enterocyte effacement (LEE) pathogenicity island, which encodes the ability to induce attaching and effacing lesions on the host intestinal mucosa. While LEE is essential for colonization of the host in these pathogens, strains of EHEC that do not carry LEE are regularly isolated from patients with severe disease, although little is known about the way these organisms interact with the host epithelium. In this study, we compared the adherence properties of clinical isolates of LEE-negative EHEC with those of LEE-positive EHEC O157:H7. Transmission electron microscopy revealed that LEE-negative EHEC O113:H21 was internalized by Chinese hamster ovary (CHO-K1) epithelial cells and that intracellular bacteria were located within a membrane-bound vacuole. In contrast, EHEC O157:H7 remained extracellular and intimately attached to the epithelial cell surface. Quantitative gentamicin protection assays confirmed that EHEC O113:H21 was invasive and also showed that several other serogroups of LEE-negative EHEC were internalized by CHO-K1 cells. Invasion by EHEC O113:H21 was significantly reduced in the presence of the cytoskeletal inhibitors cytochalasin D and colchicine and the pan-Rho GTPase inhibitor compactin, whereas the tyrosine kinase inhibitor genistein had no significant impact on bacterial invasion. In addition, we found that EHEC O113:H21 was invasive for the human colonic cell lines HCT-8 and Caco-2. Overall these studies suggest that isolates of LEE-negative EHEC may employ a mechanism of host cell invasion to colonize the intestinal mucosa.
INTRODUCTION
Infection with enterohemorrhagic Escherichia coli (EHEC) can result in a variety of clinical conditions, ranging in severity from mild diarrhea to bloody diarrhea and the hemolytic uremic syndrome (40). Strains of EHEC that carry the locus of enterocyte effacement (LEE) are associated with progression to severe disease and exhibit a distinctive mechanism of colonization that is characterized by intimate bacterial adherence to the host intestinal mucosa and the formation of attaching and effacing (A/E) lesions (20). The ultrastructural changes characteristic of A/E lesions result from cytoskeletal rearrangements in the host cell, in particular the recruitment of filamentous actin beneath adherent bacteria (28). This ability to manipulate the host cytoskeleton and induce intimate bacterial attachment is encoded by the LEE pathogenicity island of A/E pathogens such as EHEC and the closely related pathogen enteropathogenic E. coli (EPEC) (36). In these pathogens LEE is essential for colonization of the host and virulence (32, 37, 48, 51).
Throughout infection A/E pathogens remain largely restricted to the mucosal surface, and they are generally regarded as extracellular pathogens. In contrast, other enteric pathogens, such as Yersinia, Shigella, and Salmonella spp., colonize the host by invading the intestinal mucosa. Salmonella and Shigella spp. exploit the process of macropinocytosis to effect host cell entry and rely on a functional host cell cytoskeleton as well as activation of members of the Rho family of GTPases to stimulate their uptake (1, 9). The activation of Cdc42 and Rac induces host cell cytoskeletal rearrangements and membrane ruffling that results in engulfment and internalization of the bacteria (21, 38). This activation is mediated by homologous bacterial secreted effector proteins that are translocated into the host cell by type III secretion systems (21). Inhibitors of Rho GTPase activity and cytoskeletal inhibitors of actin filament formation block Salmonella- and Shigella-induced invasion (17, 55). In contrast, invasin-mediated uptake of the enteropathogenic Yersinia spp. by epithelial cells depends on high-affinity interaction between the bacterial outer membrane protein invasin and host cell 1-integrins (25). Clustering of the host cell receptor and the subsequent activation of focal adhesion kinase by tyrosine phosphorylation lead to internalization of the adherent bacteria in a process similar to receptor-mediated endocytosis (2, 53). Like that by Salmonella and Shigella, invasion by Yersinia is blocked by inhibitors of actin filament formation (57). However, whereas the tyrosine kinase inhibitor genistein blocks invasion by Yersinia, it has no effect on the internalization of Salmonella by epithelial cells (47, 52). These organisms therefore exploit distinct host cell processes to stimulate their uptake.
Although the majority of EHEC strains isolated from patients are A/E pathogens, including the O157:H7 serotype, some isolates of EHEC do not carry LEE and are not A/E pathogens (44). These strains have been termed LEE-negative or eae-negative EHEC or shiga toxigenic E. coli, and they have been regularly associated with sporadic cases and small outbreaks of hemorrhagic colitis and the hemolytic uremic syndrome worldwide (6, 16, 23, 27, 43). An early study investigating the adherence mechanisms of LEE-negative EHEC O113:H21 showed that in contrast to EHEC O157:H7, EHEC O113:H21 adhered to HEp-2 human epithelial cells without actin accumulation or A/E lesion formation (15). Transmission electron microscopy of infected HEp-2 cells revealed that the bacteria attached to the epithelial cell surface in abundance and that some bacteria were located within intracellular vacuoles. More recent studies have shown that EHEC O113:H21 also adheres to Henle 407 and Chinese hamster ovary (CHO-K1) cells (14, 42) and that LEE-negative EHEC O91:H21 adheres to human intestinal T84 cells and has the capacity to colonize the intestinal tracts of mice and cattle (13, 31, 49). Two putative adhesins have been identified that promote attachment to host cells in tissue culture. These include Saa, an autoagglutinating adhesin similar to YadA from Yersinia spp., which is encoded by the large hemolysin plasmid pO113, and LPFO113, a putative fimbrial adhesin encoded by a chromosomal gene cluster (14, 42). The aim of this work was to continue characterization of the LEE-negative EHEC adherence phenotype and compare the interactions of EHEC O113:H21 and EHEC O157:H7 with CHO-K1 cells and the human colonic cell lines HCT-8 and Caco-2.
MATERIALS AND METHODS
Bacterial strains and growth conditions. The bacterial strains used in this study are listed in Table 1. E. coli strains were grown in Luria-Bertani (LB) broth and incubated aerobically with shaking at 37°C overnight. EHEC strains EH5, EH42, and EH71 were tested for the production of EHEC hemolysin by growth on agar containing washed sheep erythrocytes, and zones of lysis were assessed at 8 h and after overnight incubation at 37°C. Stx production and stx typing were performed as described previously (4).
Cell lines and cell culture. CHO-K1 cells were maintained in alpha modified Eagle's medium (Invitrogen/Gibco, Carlsbad, CA) supplemented with 10% fetal calf serum (FCS) and 10 mM HEPES. Human colonic HCT-8 cells were maintained in RPMI 1640 (Invitrogen/Gibco) supplemented with 10% FCS, 1 mM sodium pyruvate, 2 mM L-glutamine, and 10 mM HEPES. The Caco-2 cell line was maintained as undifferentiated cells in Dulbecco's modified Eagle's medium (ThermoTrace, Noble Park, Victoria, Australia) containing 20% FCS, 9 mM sodium bicarbonate, 2 mM L-glutamine, 20 mM HEPES, 40 μg/ml gentamicin, and 2 μg/ml amphotericin B. All cell lines were incubated at 37°C in the presence of 5% CO2.
Quantitative invasion assays and eukaryotic cell inhibitors. Quantitative assessment of bacterial invasion of CHO-K1, HCT-8, and Caco-2 cells was done as described previously (46). Briefly, washed semiconfluent cell monolayers were infected in the presence of 0.5% mannose with around 107 CFU of each bacterial test strain for 3 h, after which some cell monolayers were washed five times with phosphate-buffered saline (PBS) and lysed in 0.1% digitonin. Following cell lysis, bacteria were resuspended in LB broth and quantified by plating serial dilutions to give the total number of cell-associated bacteria. To obtain the number of intracellular bacteria, a second set of infected wells was washed five times and incubated with 100 μg/ml gentamicin for 60 min. Following this incubation period, cells were washed five times with PBS, lysed with 0.1% digitonin, and resuspended in LB broth for quantification by plating serial dilutions. Assays were carried out in duplicate, and the results from at least three independent experiments were expressed as the percentage of the total cell-associated bacteria that were intracellular (mean ± standard deviation). Differences in invasion were assessed for significance by using an unpaired, two-tailed t test.
To assess the effect of eukaryotic cell inhibitors on bacterial invasion, cell monolayers were incubated with cytochalasin-D (1 μM), colchicine (1.25 μM), compactin (25 μM), or genistein (50 μM) (Sigma-Aldrich, St. Louis, MO) prior to bacterial inoculation as described previously (8, 33). Cytochalasin-D and colchicine were added to cells 30 min prior to inoculation, genistein 15 min prior to inoculation, and compactin 18 h prior to inoculation. All of the inhibitors were present throughout the 3-h infection period.
Electron microscopy. CHO-K1 cells were infected with EHEC O113:H21 strain EH41 or EHEC O157:H7 strain EDL933 for 3 h and fixed in 2.5% glutaraldehyde, followed by postfixation in 2.5% osmium tetroxide for 1 h. Following dehydration in a graded acetone series, the cell pellet was embedded in Epon-Araldite epoxy resin. Thin (0.5-μm) sections were stained with 10% uranyl acetate and 2.5% lead citrate before viewing under a Phillips CM12 electron microscope at 60 kV.
Antibodies and immunofluorescence microscopy. To visualize EHEC O113:H21 for immunofluorescence studies, rabbit polyclonal antibodies were raised to surface components of EHEC O113:H21. Briefly, supernatant extracts were collected by 10% trichloroacetic acid precipitation from EHEC O113:H21 EH41 grown overnight in Dulbecco's modified Eagle's medium; 100 μg of the precipitate was resuspended in PBS and Freund's complete adjuvant and used to immunize a rabbit by intramuscular inoculation. Primary immunization was followed by three booster injections at 3-week intervals using Freund's incomplete adjuvant, and the resulting antiserum was absorbed three times with whole heat-killed E. coli DH5.
For immunofluorescence, approximately 105 CHO-K1 cells or HCT-8 cells were grown on 12-mm-diameter coverslips in 24-well plates (Sarstedt, Nümbrecht, Germany). Wells were inoculated with around 5 x 107 CFU of each test strain and incubated for 5, 15, or 30 min at 37°C in 5% CO2. Following the incubation period, cells were washed five times and fixed with 10% formalin for 20 min. To visualize extracellular bacteria, coverslips were incubated at room temperature for 60 min with anti-O113:H21 diluted 1:100 in PBS containing 0.2% bovine serum albumin (PBS-BSA). Coverslips were washed three times and incubated for 60 min with a second anti-rabbit antibody conjugated to tetramethylrhodamine isothiocyanate (TRITC) (Sigma) diluted 1:200 in PBS-BSA. Following this initial staining, cells were permeabilized with 0.1% Triton X-100 for 4 min and washed three times with PBS. To stain intracellular bacteria, anti-O113:H21 antibodies were diluted 1:100 in PBS-BSA and incubated with coverslips for 60 min as before. Coverslips were then washed three times and incubated with a second anti-rabbit antibody conjugated to fluorescein-6-isothiocyanate (FITC) (Sigma) diluted 1:200 in PBS-BSA. To visualize the cell cytoskeleton, permeabilized cells were incubated with phalloidin-TRITC (0.05 μg/ml) (Sigma). All preparations were examined by epifluorescence microscopy using an Olympus BX51 microscope with a 100x oil immersion objective (Olympus, Tokyo, Japan). Digital images were acquired using an Olympus DP-70 digital camera and merged using DP manager software version 1.1.1.71.
Construction of an escF deletion mutant of EDL933. To construct an escF mutant of EDL933, the deleted escF region carrying a kanamycin resistance marker was amplified by PCR from EPEC escF ICC171 (56) by using the primers 5'-CACTGACTCGTTTGCTCG-3' and 5'-CTAATCTTGGCTAACTCTC-3' under the following conditions: 2 min at 94°C; 30 cycles of 44 s at 94°C, 40 s at 46°C, and 1 min at 72°C; and an extension period of 5 min at 72°C. The resulting PCR product was introduced into EDL933 harboring pKD46 and recombined into the EDL933 genome as described previously (12).
Detection of known invasion genes by PCR and low-stringency hybridization. Oligonucleotide primers were designed to amplify inv from Yersinia enterocolitica W22703 (5'-TGCGTACCTTCCAACAAAG-3' and 5'-GGCGTACTATCAATATTAGTC-3', covering nucleotides 439 to 1441 of the native gene), sipC from Salmonella enterica serovar Typhimurium LT2 (5'-GAGTCCTACACTGAGCGC-3' and 5'-TTGCCTGCGATAGCAGCG-3'), and ipaC from enteroinvasive E. coli (EIEC) 6/84 (5'-CAGCAGATTGCAGCGCATAT-3' and 5'-CAAGAGCAGATGCATAACGC-3') by PCR. PCR amplification was performed on 500 ng of template DNA with approximately 1 μg of each primer PCR per 100-μl reaction mixture. Amplification was performed under the following conditions: 2 min at 94°C; 30 cycles of 44 s at 94°C, 40 s at the appropriate annealing temperature (inv, 50°C; sipC, 45°C; ipaC, 50°C), and 1 min at 72°C; and an extension period of 5 min at 72°C. PCR-generated DNA products were examined by agarose gel electrophoresis. To generate digoxigenin-labeled probes, digoxigenin-dUTP was incorporated into the PCR according the manufacturer's instructions (Roche, Indianapolis, IN). Southern hybridizations were performed at low stringency (55 to 60°C) according to the manufacturer's protocol (Roche). Genomic DNA for hybridizations and PCR was isolated using the DNeasy tissue kit (Qiagen, Hilden, Germany) according the manufacturer's instructions.
RESULTS
Interaction of EHEC O113:H21 and EHEC O157:H7 with CHO-K1 cells. Previously we reported that EHEC O113:H21 strain EH41 adheres to CHO-K1 epithelial cells (14). In this study we examined this interaction more closely by comparing the adherence properties of LEE-negative EHEC with those of LEE-positive EHEC O157:H7. CHO-K1 cells were incubated with EHEC O113:H21 strain EH41 and EHEC O157:H7 strain EDL933 for 3 h and examined by transmission electron microscopy. Unexpectedly, this showed that EHEC O113:H21 was internalized and that the bacteria were frequently located within a membrane-bound vacuole (Fig. 1A and inset). Although extracellular bacteria were also seen in association with EH41-infected cells, these were located with cells that had undergone necrosis (Fig. 1C). In contrast, EHEC O157:H7 strain EDL933 remained extracellular, intimately attached to the epithelial cell surface (Fig. 1B), and extracellular bacteria were not associated with necrotic cells (Fig. 1D).
Ability of EHEC O113:H21 and other serotypes of LEE-negative EHEC to invade CHO-K1 cells. To determine if other clinical isolates of EHEC O113:H21 and other serotypes of LEE-negative EHEC were capable of invading epithelial cells, we performed quantitative invasion assays on CHO-K1 cells infected with various strains of EHEC (46). The invasive capacities of various strains and serotypes of LEE-negative EHEC were compared with those of two isolates of EIEC (which exhibit a Shigella-like mechanism of invasion), two isolates of EHEC O157:H7, and one isolate of EPEC (strain E2348/69, which has previously been reported to invade epithelial cells [19, 26]). Noninvasive E. coli HB101 and E. coli DH5 served as negative controls. All strains of LEE-negative EHEC expressed EHEC hemolysin and Stx-2, as determined previously (4, 16) or in this study. The quantitative invasion assays showed that isolates of LEE-negative EHEC were significantly more invasive for CHO-K1 cells than EHEC O157:H7, EPEC E2348/69, and E. coli HB101 and DH5 (Fig. 2). These data confirmed that EHEC O113:H21 was able to invade epithelial cells and showed that the levels of CHO-K1 cell invasion by all LEE-negative EHEC strains irrespective of serotype were comparable to those observed for EIEC strains (Fig. 2). In addition, we tested the ability of a pO113-cured derivative of EHEC O113:H21, strain EH41c, to invade epithelial cells. Plasmid-borne invasins have been well characterized for other pathogens, such as Shigella, and pO113 encodes several putative virulence determinants, including Saa, which shares similarity with the plasmid-encoded adhesin/invasin YadA from Y. enterocolitica. There was no difference, however, in the levels of invasion observed for EH41 and EH41c, indicating that pO113-encoded factors were not required to induce EH41 uptake by CHO-K1 cells, although this does not necessarily discount a role for the large hemolysin plasmid in other LEE-negative invasive strains. In addition, to account for the possibility that EH41 and EDL933 share invasion genes but that the LEE pathogenicity island contributes to an antiphagocytic effect which would result in decreased invasion, we tested the ability of an escF deletion mutant of EHEC O157:H7 EDL933 to invade CHO-K1 cells. However, there was no difference in invasion levels between wild-type EDL933 and the escF mutant strain (data not shown), indicating that LEE does not interfere with EHEC O157:H7 uptake.
Kinetics of EHEC O113:H21 invasion of CHO-K1 cells. The quantitative gentamicin protection assay used in this study indicated the number of intracellular bacteria at 3 h postinoculation. To examine the early stages of epithelial cell invasion by EHEC O113:H21, we used an immunofluorescence assay to differentiate between intracellular and extracellular bacteria at 5, 15, and 30 min postinfection. A similar approach has been used previously to examine invasion by Salmonella (50). Following incubation with bacteria, CHO-K1 cells were fixed and incubated with anti-O113:H21 antibodies and secondary antibodies conjugated to TRITC or FITC. This procedure labeled intracellular bacteria with FITC, while extracellular bacteria were labeled with both TRITC and FITC. Therefore, intracellular bacteria fluoresced green, while extracellular bacteria fluoresced yellow/orange due to colocalization of TRITC and FITC. In addition the cell cytoskeleton was stained with phalloidin-TRITC. The results showed that 5 min after inoculation, some bacteria that were associated with the host cell were located intracellularly, although the majority appeared to be surface located (Fig. 3, upper left panel). Extracellular bacteria were also visible by phase-contrast microscopy (data not shown). At 15 min and 30 min the majority of bacteria fluoresced green, indicating that invasion was largely complete (Fig. 3, upper right and lower left panels). Intracellular bacteria were not visible by phase-contrast microscopy (data not shown). Interestingly, at all time points some bacteria were observed to stain partly green and partly orange, suggesting these organisms were in the process of cell invasion (Fig. 3, lower right panel). This apparent cell entry was not associated with any obvious ruffling of the host cell membrane or focusing of the actin cytoskeleton. Again, the extracellular component of the invading bacterium was visible by phase-contrast microscopy (data not shown).
Effect of eukaryotic cell inhibitors on EHEC O113:H21 invasion of CHO-K1 cells. Many invasive pathogens exploit specific host cell pathways to induce their own uptake. To identify host cell structures and processes that were involved in epithelial cell invasion by LEE-negative EHEC, we treated CHO-K1 cells with eukaryotic cell inhibitors known to block bacterial invasion by various pathogens (8, 11, 33, 47). CHO-K1 cells were treated with cytochalasin D to disrupt actin microfilament formation or with colchicine to inhibit microtubule function and subsequently infected with EHEC O113:H21 EH41. Treatment with either of these agents blocked CHO-K1 cell invasion by strain EH41, demonstrating that a functional host cell cytoskeleton is needed for bacterial uptake (Table 2). In contrast, bacterial adherence was unaffected by the inhibitors (data not shown). To ascertain if any members of the Rho family of GTP-binding proteins played a role in internalization of EH41, CHO-K1 cells were treated with the pan-Rho GTPase inhibitor compactin. Bacterial invasion of compactin-treated CHO-K1 cells was significantly reduced compared with that of untreated cells, indicating that Rho GTPases were important for EH41-mediated internalization (Table 2). In contrast, the treatment of CHO-K1 cells with genistein had no significant impact on invasion by strain EH41 (Table 2). Again, neither inhibitor affected levels of bacterial adherence (data not shown).
Invasion of HCT-8 and Caco-2 cells by EHEC O113:H21. Although CHO-K1 cells are widely used to study bacterial interactions with epithelial cells, they do not represent a host cell type that is particularly relevant to human infection. Therefore, to ensure that invasion by EHEC O113:H21 uptake was not peculiar to CHO-K1 cells, we also infected the human colonic cell lines HCT-8 and Caco-2 with EHEC O113:H21 strain EH41. Quantitative gentamicin protection assays showed that EH41 and EIEC strain 6/84 were significantly more invasive for HCT-8 cells than EDL933 and E. coli HB101 (Table 3). As for CHO-K1 cells, the level of EH41 invasion into HCT-8 cells was not significantly different from that of EIEC 6/84. In addition, EH41 was significantly more invasive for Caco-2 cells than EHEC EDL933 and E. coli HB101 (Table 3). These results demonstrated that LEE-negative EHEC strains were invasive for epithelial cells of human intestinal origin.
The level of E. coli HB101 internalization into HCT-8 cells was surprisingly high compared with invasion levels observed in CaCo-2 or CHO-K1 cells (Fig. 1 and Table 3). We therefore repeated the quantitative invasion assays in HCT-8 and CHO-K1 cells with a strain of E. coli DH5 as a further negative control (Fig. 1 and Table 3). The results showed that while E. coli DH5 invasion in CHO-K1 cells was equivalent to that of E. coli HB101 (0.35% ± 0.26% and 0.11% ± 0.08%, respectively), in HCT-8 cells, E. coli HB101 invasion was significantly higher than that of E. coli DH5 (P = 0.034) (Table 3). This suggested that the strain of E. coli HB101 used in this study showed an unusual capacity to invade HCT-8 cells but not CaCo-2 or CHO-K1 cells. However, similar to the case for E. coli HB101, the level of E. coli DH5 internalization in HCT-8 cells was higher than that in CHO-K1 cells (1.2% ± 0.69% and 0.35% ± 0.26%, respectively), suggesting that the HCT-8 cell line also has a greater inherent capacity to take up bacteria than CHO-K1 or CaCo-2 cells.
Screening for known enterobacterial invasion determinants. As no genome sequence is available for EHEC O113:H21 or any LEE-negative EHEC strain, we tested for the presence of known invasion genes of enterobacteria by PCR and low-stringency Southern and dot blot hybridizations. The genes examined included a 5' region of inv from Y. enterocolitica, which encodes invasin, and sipC from S. enterica serovar Typhimurium and ipaC from EIEC, which encode components of their respective type III secretion systems that are essential for inducing host cell ruffling and bacterial uptake. No homologous nucleotide sequences were detected for any of these genes by PCR in the LEE-negative EHEC strains, but low-stringency hybridization revealed that three of the eight invasive strains tested, EH5, EH53, and EH71, carried sequences homologous to ipaC although not to sipC or inv (data not shown).
DISCUSSION
Attachment to the intestinal mucosa by EHEC is an essential step towards colonization of the host and the development of disease. Most clinical isolates of EHEC are A/E pathogens that carry the LEE pathogenicity island and induce well-characterized and distinctive lesions on the intestinal epithelium following intimate adherence to the enterocyte surface. However, LEE-negative EHEC strains are not A/E pathogens, and in the absence of LEE, little is known about how these strains colonize the host (44). In this study we were able to show that, in contrast to EHEC O157:H7, various serotypes of LEE-negative EHEC were invasive for CHO-K1 epithelial cells. Although EHEC O157:H7 has been reported to enter cells at a higher rate than E. coli HB101 (41), which was also true in our study, here invasion by LEE-negative EHEC strains was consistently 5- to 10-fold higher than invasion by two isolates of EHEC O157:H7. The level of invasion observed for LEE-negative EHEC strains was also significantly higher than that observed for EPEC E2348/69 and matched that of two EIEC isolates which exhibit a Shigella-like mechanism of internalization. However, unlike Shigella and EIEC, which escape from the phagosome into the cell cytoplasm, EHEC O113:H21 remained within a membrane-bound vacuole. Investigation of the kinetics of CHO-K1 cell invasion by EHEC O113:H21 showed that internalization was rapid and occurred within minutes of bacterial attachment to the cell surface. The LEE-negative EHEC invasion phenotype was also observed in the human colonic cell lines HCT-8 and Caco-2, which represent a more relevant in vitro model of infection than CHO-K1 cells. Interestingly, the level of bacterial invasion into HCT-8 cells for all strains tested was somewhat higher than the levels observed for CHO-K1 cells and Caco-2 cells, suggesting that HCT-8 cells have some inherent capacity for phagocytosis.
Many bacterial pathogens, including enteric pathogens, invade mucosal surfaces in order to colonize the host. Invasion of the intestinal mucosa is a feature of infections by Yersinia, Salmonella, and Shigella spp. Uptake of the bacteria by host cells relies on a functional host cell cytoskeleton in all cases (21). Using the cytoskeletal inhibitors cytochalasin-D and colchicine, we showed that invasion of CHO-K1 cells by EHEC O113:H21 required an intact actin cytoskeleton as well as microtubule function. Although microtubule function is not required for invasion by Salmonella, Shigella, and Yersinia (18), microtubules have been shown to be important for the uptake of other invasive pathogens (5, 8, 10, 11, 24). In addition, use of the inhibitors compactin and genistein showed that EHEC O113:H21 invasion relied upon the activity of Rho GTPases but not on that of tyrosine kinases.
Rho GTPases regulate cytoskeletal rearrangements within host cells (22). In response to contact with Salmonella spp., actin ruffling of the host cell surface, which coincides with bacterial invasion, is driven by activation of Cdc-42 and Rac. The zipper mechanism of host cell invasion by Yersinia spp., on the other hand, requires tyrosine kinase activity as well as activation of Rho GTPases, in particular Rac1 (3, 25). While the results of our experiments with inhibitors of bacterial uptake suggested that invasion by EHEC O113:H21 depended upon the same cellular processes as Salmonella-mediated uptake, we were unable to identify any membrane ruffling associated with EHEC O113:H21 entry into CHO-K1 cells by using phalloidin to stain for filamentous actin. Martinez and Hultgren have described the involvement of Rho-GTPases in the zipper mechanism of invasion of bladder epithelial cells by uropathogenic E. coli, which invades using type I pili (33), but this process also requires tyrosine kinase activity (34). Hence, the precise pathway of LEE-negative EHEC-induced entry is still unclear.
At this stage the genetic basis of invasion by LEE-negative EHEC is unknown. The inclusion of mannose in our invasion assays precluded the involvement of type I pili as described for uropathogenic E. coli and adherent-invasive E. coli, which is associated with Crohn's disease (7, 34), and although several virulence-associated factors have been identified on the large hemolysin plasmid pO113, curing of this plasmid from EHEC O113:H21 strain EH41 had no impact on CHO-K1 cell invasion, implying that the putative invasins are likely to be chromosomally encoded, at least in strain EH41. We also screened for homologues of known invasion genes, including inv, ipaC, and sipC. The presence of a putative ipaC homologue in some invasive LEE-negative EHEC strains but not others may indicate that these pathogens carry diverse invasion loci, some of which may be related to the ipa invasion locus of EIEC. Identification of the ipaC homologue in strains EH5, EH53, and EH71 would help to characterize the genetic basis of invasion in these strains, and further testing will show how prevalent this locus is among LEE-negative EHEC isolates. Nevertheless, since five of the eight LEE-negative EHEC strains tested in this study were negative for the ipaC probe, it is likely that other, as-yet-unidentified genes contribute to the invasion phenotype. Overall this study suggests that LEE-negative EHEC strains may employ a mechanism of host cell invasion to colonize the intestinal epithelium which in the pathogenesis of these infections may compensate for the absence of LEE and the inability to form A/E lesions.
ACKNOWLEDGMENTS
We are indebted to James Paton and Adrienne Paton, University of Adelaide, for the gift of the HCT-8 cell line and to Carl Kirkwood, Murdoch Children's Research Institute, for the CaCo-2 cell line. We are also grateful to Gadi Frankel, Imperial College, London, for sending us strain ICC171. We greatly appreciate the assistance of Julia Prentice in the construction of the escF deletion mutant of EDL933, and we thank Yan Tan for culturing the CaCo-2 cell line.
This work was supported by the Australian National Health and Medical Research Council.
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