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Infection and Immunity, January 2009, p. 517-531, Vol. 77, No. 1
0019-9567/09/$08.00+0     doi:10.1128/IAI.00695-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Human Decay-Accelerating Factor and CEACAM Receptor-Mediated Internalization and Intracellular Lifestyle of Afa/Dr Diffusely Adhering Escherichia coli in Epithelial Cells{triangledown}

Julie Guignot, Sylvie Hudault, Imad Kansau, Ingrid Chau, and Alain L. Servin*

INSERM, UMR756, Signalisation et Physiopathologie des Cellules Epithéliales, Châtenay-Malabry, France, and Université Paris-Sud 11, Faculté de Pharmacie, Châtenay-Malabry, France

Received 3 June 2008/ Returned for modification 18 July 2008/ Accepted 2 November 2008


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ABSTRACT
 
We used transfected epithelial CHO-B2 cells as a model to identify the mechanism mediating internalization of Afa/Dr diffusely adhering Escherichia coli. We provide evidence that neither the {alpha}5 or β1 integrin subunits nor {alpha}5β1 integrin functioned as a receptor mediating the adhesion and/or internalization of Dr or Afa-III fimbria-positive bacteria. We also demonstrated that (i) whether or not the AfaD or DraD invasin subunits were present, there was no difference in the cell association and entry of bacteria and that (ii) DraE or AfaE-III adhesin subunits are necessary and sufficient to promote the receptor-mediated bacterial internalization into epithelial cells expressing human decay-accelerating factor (DAF), CEACAM1, CEA, or CEACAM6. Internalization of Dr fimbria-positive E. coli within CHO-DAF, CHO-CEACAM1, CHO-CEA, or CHO-CEACAM6 cells occurs through a microfilament-independent, microtubule-dependent, and lipid raft-dependent mechanism. Wild-type Dr fimbria-positive bacteria survived better within cells expressing DAF than bacteria internalized within CHO-CEACAM1, CHO-CEA, or CHO-CEACAM6 cells. In DAF-positive cells, internalized Dr fimbria-positive bacteria were located in vacuoles that contained more than one bacterium, displaying some of the features of late endosomes, including the presence of Lamp-1 and Lamp-2, and some of the features of CD63 proteins, but not of cathepsin D, and were acidic. No interaction between Dr fimbria-positive-bacterium-containing vacuoles and the autophagic pathway was observed.


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INTRODUCTION
 
Diffusely adhering Escherichia coli (DAEC) organisms comprise two classes of strains, the typical DAEC and the atypical DAEC strains, each subdivided into two subclasses of strains (67). These pathogenic E. coli strains belong to group six of enterovirulent E. coli (38). Typical Afa/Dr DAEC strains have been shown to be involved in age-dependent diarrhea in infants (48, 63). Typical Afa/Dr DAEC strains have been shown to belong to the recently reported type IV pathotype of uropathogenic E. coli (UPEC) (46). Typical Afa/Dr DAEC strains are involved in urinary tract infections (UTIs), since 25 to 50% of children with cystitis and 30% of pregnant women with pyelonephritis are infected with E. coli bearing Afa/Dr fimbriae (30, 57). Moreover, typical Afa/Dr DAEC strains are involved in recurrent UTIs, and the vast majority of the typical Afa/Dr DAEC isolates (90%) are multiantibiotic resistant (29). The typical Afa/Dr DAEC strain expresses a family of genes that is organized to form a family of afa-, dra-, daa-, and/or nfa-related operons encoding Afa-I, Afa-II, Afa-III, Afa-V, Dr, Dr-II, F1845, and Nfa-I fimbriae (67). The genes are organized in similar ways, with at least five genes (A to E), of which the last, the E gene, encodes a major structural adhesin subunit (70). The D gene encodes the invasin subunit (21, 35, 72). Importantly, DraE and AfaE-III proteins display 98% sequence identity, whereas DraD and AfaD-III share 100% sequence identity. Atomic resolution models of Dr and Afa-III fibrils have revealed that the structural basis for assembly occurs by donor strand complementation and that the architecture of capped surface fibers results from the assembly of several DraE or AfaE subunits, with one invasin subunit, DraD or AfaD-III, at their distal ends (1, 14).

Typical Afa/Dr fimbriae govern the adhesion of the bacteria to host epithelial cells and the cells' responses to their presence. All these fimbriae (Afa/DrDAF) are able to bind specifically to the complement control protein repeats 2 and 3 (CCP2 and CCP3), domains of human decay-accelerating factor (DAF; CD55) (56). The DAF binding domain into the Afa/Dr adhesin subunit is located in its central part, localized on strands B and E of DraE (1, 31). Moreover, our group has recently reported that a subclass of Afa/Dr fimbriae, including Afa-III, Dr, and F1845 (Afa/DrCEACAM), recognized members of the human CEACAM family (3), which includes CEACAM1 (biliary glycoprotein; CD66a), CEA (carcinoembryonic antigen; CD66e), and CEACAM6 (nonspecific cross-reacting antigen; CD66c) (4, 27). AfaE-I, AfaE-III, AfaE-V, DraE, and DaaE adhesin subunits of Afa/Dr DAEC targeted the N-terminal domains of CEACAMs (40). The CEACAM binding site is located primarily in the A, B, E, and D strands of the Dr adhesin opposite the beta-sheet encompassing the previously determined binding site for DAF (40). Typical Afa/Dr DAEC strains have been described as invasive in unpolarized epithelial cells expressing several Afa/Dr fimbriae receptors but with a low level of efficiency (24, 26, 35). In cultured human polarized intestinal cells forming a cell monolayer that mimics an epithelium, these bacteria are apically uninvasive and enter the cells via the basolateral domain (26). The process of internalization into unpolarized epithelial cells involves lipid rafts (26, 37, 66) and dynamic, unstable microtubules (24, 26). Previous studies conducted with Dr and Afa-III fimbriae have not elucidated the processes by which typical Afa/Dr DAEC strains enter epithelial cells, which remain controversial. Two different working hypotheses have been proposed. According to one possible mechanism, after the Dr fimbriae have recognized the membrane-bound human DAF, the entire dra operon is necessary to trigger a receptor-mediated internalization of the bacteria (24). Selvarangan et al. (66) have shown that a transposon mutant with a mutation in the DraE adhesin subunit lacks adhesiveness and consequently fails to enter the cells. More convincingly, Das et al. (15) have demonstrated that the DraE subunit is both an adhesin and an invasin, since by mutagenesis to replace selected amino acids in hydrophilic domain II of the DraE protein, bacterial internalization was reduced or abolished without modifying the bacterial cell association. The second possible mechanism would imply that DraD and AfaD-III invasin subunits recognize the membrane-bound {alpha}5β1 integrin, and this is sufficient to trigger the entry of bacteria (26, 61) via a zipper-like mechanism (37). For this second mechanism, it has not been clearly established whether there is an initial step of recognition of the membrane-bound receptor DAF or CEACAM members by the AfaE/DraE adhesin subunits.

We decided to conduct a series of experiments to analyze the role of DAF and/or {alpha}5β1 integrin in the receptor-mediated internalization of Afa/Dr DAEC. We extended our investigation by analyzing the role of the CEACAMs that act as receptors for Afa/DrCEACAM adhesins during the receptor-mediated internalization of Afa/Dr DAEC. We also investigated the role of AfaE/DraE adhesins and/or AfaD/DraD invasin subunits in bacterial internalization. We also examined the intracellular lifestyle of Dr fimbria-positive bacteria by comparing the survival rates of the intracellular bacteria in a series of cell lines, each of which expresses one of the membrane-bound epithelial receptors of Afa/Dr fimbriae, and by characterizing the late vacuole-containing internalized bacteria in a DAF-positive epithelial cell line.


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MATERIALS AND METHODS
 
Antibodies and reagents. Sodium butyrate, saponin, rapamycin, nocodazole, cytochalasin D, methyl-β-cyclodextrin, bovine serum albumin, collagen, and fibronectin were obtained from Sigma (Sigma-Aldrich Chimie SARL, L'Isle d'Abeau Chesnes, France). Hygromycin and puromycin were from InvivoGen (Cayla SA, Toulouse, France). G418 (Geneticin) was from PAA Laboratories (Les Mureaux, France).

Anti-Lamp-1 (lysosome-associated membrane protein 1; clone H4A3), Lamp-2 (clone H4B4), and CD63 (clone H5C6), developed by J. T. August and J. E. Hildreth (The Johns Hopkins University School of Medicine, Baltimore, MD) (45), were used at a dilution of 1:200. They were obtained from the Developmental Studies Hybridoma Bank developed under the auspices of NICHD and maintained by the University of Iowa (Department of Biological Sciences, Iowa City, IA). Rabbit anti-cathepsin D polyclonal antibody (PAb) was from J. S. Mort (McGill University, Montreal, Canada) and was used at a dilution of 1:50. Mouse anti-DAF monoclonal antibody (MAb) 8D11 was from D. M. Lublin (Washington University School of Medicine, St. Louis, MO) and was used at dilution of 1:100. Mouse anti-CEACAM antibody (clone D14HD11) that recognizes CEACAM1, -3, -4, -5, and -6 was from Genovac (Aachen, Germany) and was used at a dilution of 1:100. Anti-{alpha}5 integrin subunit (Immunotech SA, Marseille, France) and anti-β1 integrin subunit (clone MAR4; BD Biosciences, Erembodegem, Belgium) were used at a dilution of 1:500 and 1:100, respectively. Anti-β1 antibody (clone P5D2), developed by E. A. Wagner (Hutchinson Cancer Research Center, Seattle, WA), was obtained from the Developmental Studies Hybridoma Bank and was used at a dilution of 1:100. Texas Red sulfonyl chloride-conjugated, cyanine 5-conjugated, and fluorescein isothiocyanate-conjugated donkey anti-mouse and anti-rabbit antibodies were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA) and used at a dilution of 1:400. Mouse peroxidase-conjugated antibody (Amersham Pharmacia Biotech, Inc., Orsay, France) was used at a dilution of 1:100. LysoTracker was from Molecular Probes (Invitrogen, Cergy, France).

Bacterial strains. The bacterial strains used in this study are listed in Table 1. Stock cultures were maintained on 30% glycerol at –80°C. Before the experiments, the bacterial strains were transferred onto fresh Luria-Bertani (LB) agar (Difco Laboratories, Detroit, MI) and incubated at 37°C for 18 h. For each experiment, bacteria were subcultured in LB broth at 37°C for 18 h. On the day of the experiment, bacteria were washed twice with sterile phosphate-buffered saline (PBS) and recovered using appropriate tissue culture media (Invitrogen).


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  		TABLE 1. Bacterial strains used in this study

Disruption of the draD gene into the WT-IH11128 strain. A nonpolar mutation that deletes the entire draD gene into the wild-type IH11128 (WT-IH11128) strain from the initiation to the stop codon was created by allelic exchange with the nonpolar aphaA gene using a three-step PCR procedure described previously (16). PCR fragments were used with 5'-GGTGATGCCTTATTCCAGGGTC-3' and 5'-CTTCACGAGGCAGACCTCAGCGCCTAAAGCGCAGAAGAAAAACAG-3' primers to amplify the right fragment and 5'-CAGCATGGTTGCCCACAGTGAG-3' and 5'-GATTTTGAGACACAACGTGGCTTTCATCTCCGGCTCCTCCCGTCA-3' primers to amplify the left fragment. The recombinogenic fragment carrying an antibiotic resistance gene flanked by regions homologous to the target locus has been electroporated into the WT-IH11128 strain expressing the highly proficient homologous recombination system encoded by plasmid pKOBEG-A. Isogenic mutant clones were verified by sequence analysis after sequencing was performed by Genome Express (Meylan, France) at the premium quality setting.

Site-directed mutagenesis. Site-directed mutagenesis was performed using the QuikChange site-directed mutagenesis kit (Stratagene, Agilent Technologies Company, Massy, France) to replace the lysine at residue 16 of the draD gene by a stop codon. A set of synthetic oligonucleotides (5'-GCGTGTCACCTGCGGGTAACTGATGGTCATGAGTGGTG-3' and 5'-CACCACTCATGACCAGTTACATCCCGCAGGTGACACGC-3') was used to produce the AAEC185D–E+ strain.

Preparation of Dr PAb. An antibody against the DraE protein was generated after cloning the draE gene into the pQE30 vector. The Hexa-His-DraE protein was purified using a Talon metal affinity resin (Takara Bio Europe/Clontech, Saint-Germain-en-Laye, France) under denaturing conditions, as previously described (20). Rabbit PAb was elicited by injecting purified proteins into rabbits (Agro-Bio, La Ferté Saint Aubin, France). Specificity was determined by immunoblotting.

Cell lines, transfections, and culture conditions. The cell lines used in this study are listed in Table 2. The {alpha}5β1 integrin-deficient CHO (Chinese hamster ovary) cell line (CHO clone B2), which expresses ~2% of wild-type {alpha}5β1 (65), was obtained from E. Craig and P. Mold (University of Manchester, United Kingdom). CHO cell lines permanently expressing the various known receptors of Afa/Dr DAEC have been obtained by transfection using the FuGENE-6 method (Roche Diagnostic, Meyland, France) according to the manufacturer's instructions. Plasmids encoding human DAF, CEACAM1, CEACAM1 Y493F, CEACAM1 Y520F, CEACAM1 R464Stop, CEA, CEACAM6, {alpha}5 integrin subunit, or β1 integrin subunit have been described previously (5, 12, 17, 62). The cDNA used to establish the CHO-CEACAM1 cell line encodes the longest splice variant, hCEACAM1-4L. Cells resistant for hygromycin (1,000 µg/ml) and/or puromycin (10 µg/ml) and/or G418 (800 µg/ml) were selected. The resistant clones were then screened for DAF, CEACAM1, CEA, CEACAM6, and {alpha}5 and β1 integrin subunit expression by an immunocytochemistry test. The transfected cells were maintained in Dulbecco's modified Eagle's medium-Ham F-12 medium (Invitrogen) supplemented with 5% fetal calf serum and containing hygromycin (400 µg/ml), puromycin (10 µg/ml), or G418 (300 µg/ml) for further routine culturing at 37°C in an atmosphere containing 5% CO2. Sodium butyrate (2.5 mM) has been widely used to culture transfected CHO-B2 cell lines to obtain high-level expression and ensure correct glycosylation of recombinant proteins (50).


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  		TABLE 2. Cell lines used in this study

HeLa-green fluorescent protein (GFP)-light chain 3 (LC3) cells stably transfected to express the GFP-labeled autophagosomal marker, microtubule-associated protein LC3 (GFP-LC3), were obtained from A. M. Tolkovsky (Department of Biochemistry, University of Cambridge, United Kingdom). HeLa and transfected HeLa-GFP-LC3 cell lines were grown in RPMI 1640 medium with L-glutamine (Invitrogen) supplemented with 5% heat-inactivated fetal calf serum (Invitrogen), nonessential amino acids (1%), and sodium pyruvate (1%) at 37°C in an atmosphere containing 5% CO2. HeLa-GFP-LC3 cells were cultured in the presence of 500 µg/ml of G418 (Invitrogen).

All cell lines were harvested from the flask using trypsin (0.5 mg/ml) and EDTA (0.2 mg/ml), washed once with medium, and seeded into culture plates (thiamine PPi; ATGC Biotechnologie, Noisy Le Grand, France) at the appropriate cell densities.

Cell attachment assay. Plates were coated with fibronectin or collagen type 1 (50 µl of protein solution/well) for 1 h at 37°C. Plates were then washed twice with PBS and blocked with 1% heat-denaturated (30 min; 80°C) bovine serum albumin. CHO or CHO-{alpha}5β1 integrin cells (2 x 105 cells per well) were seeded and allowed to attach for 1 h at 37°C. Cells were microscopically observed after Giemsa staining.

Bacterial cell infection and gentamicin protection assay. Cell monolayers and bacterial cultures were washed twice with PBS before infection. Infecting E. coli bacteria were suspended in culture medium, and a total of 108 CFU/ml of this suspension was then added to each well of the tissue culture plate. In the presence of the WT-IH11128 strain, the infection assay was conducted in the presence of 1% D-mannose to prevent type 1-pilus-mediated binding. The plates were incubated at 37°C in 5% CO2/95% air for 3 h. The monolayers were then vigorously washed four times with sterile PBS. Separate protocols were then used to quantify cell-associated bacteria and internalized bacteria.

Cell association of E. coli was determined by quantitative determination of bacteria associated with the infected cell monolayers. Cells were lysed with 1% sterile saponin. Appropriate dilutions were made in sterile PBS and then plated on LB agar to determine the number of viable cell-associated bacteria by bacterial colony counting.

Internalization of E. coli was determined by the quantitative determination of bacteria located within the infected cell monolayers using the gentamicin protection assay. Cells were incubated for 1 h in a medium containing 100 µg/ml of gentamicin. Bacteria that adhered to the cells were rapidly killed, whereas those located within the cells were not. The monolayer was washed once with PBS and lysed with 1% sterile saponin. Appropriate dilutions were plated on LB agar to determine the number of viable intracellular bacteria by bacterial colony counting.

Despite high selection pressure, over time some of the transfected cell lines ceased to express the CEACAM proteins. In order to normalize our results for each transfected cell line, the percentage of cells expressing a CEACAM protein was quantified by immunofluorescence for each experiment, and the bacterial association or invasion found was corrected to 100% expression. Results are expressed as CFU/5 x 105 cells. Each assay was conducted at least in triplicate with three successive passages of cultured cells.

Cell treatments. Rapamycin was used at 500 nM to activate autophagy (13). The working concentrations used were methyl-β-cyclodextrin (5 mM), cytochalasin D (1 µg/ml), and nocodazole (10 µM) (13, 26). Drugs were added directly to the culture medium, pretreated for 45 to 60 min prior to infection, and maintained throughout the infection period. The concentrations of inhibitors used in the experiments were not toxic to the cells (13, 26).

Intracellular survival. To measure the intracellular survival of bacteria in HeLa cells or transfected CHO cells, cell monolayers were infected for 3 h as described above. The infected cells were then incubated with gentamicin as described above to kill the extracellular adhering bacteria. The infected cells were subsequently cultured in the presence of cell culture medium containing gentamicin (15 µg/ml) to prevent extracellular replication of any remaining viable extracellular bacteria, and the medium was changed daily. The number of intracellular bacteria was determined 24, 48, 72, and 96 h postinfection.

siRNAs and transfection. Small interfering RNA (siRNA) oligonucleotides targeting human β1 integrin (siRNA ID 109879 and 109878) were designed and synthesized by Ambion, Inc. (Applied Biosystems, Courtaboeuf, France). The day before transfection, HeLa cells were seeded into 12-well plates in order to reach 60% confluence by the day of transfection. Transient transfection of 50 nM siRNAs was carried out using INTERFERin (Ozyme, Saint-Quentin-en-Yvelines, France) according to the manufacturer's instructions. After 48 h, the cells were infected as described above. Under these conditions, about 99% of the β1 integrin was silenced as confirmed by Western blot analysis.

Immunofluorescence and immunocytochemistry. For indirect immunofluorescence or immunochemistry labeling, cultured cells were prepared on glass coverslips. Cells were fixed in 3% paraformaldehyde in PBS (pH 7.4) for 15 min at room temperature and then washed three times in PBS. Antibodies were diluted in 10% horse serum, 0.1% saponin in PBS. Coverslips were washed twice in PBS containing 0.1% saponin, incubated for 1 h with primary antibodies, washed twice with 0.1% saponin in PBS, and incubated for 30 min with secondary antibodies. Coverslips were washed twice in 0.1% saponin in PBS, once in PBS, and once in H2O. For lysosome staining, cells were incubated for 30 min with 75 nM of LysoTracker.

GFP-WT-IH11128 bacteria were used to track the localization of intracellular bacteria. After being fixed with 3% paraformaldehyde, extracellular GFP-expressing bacteria were labeled under unpermeabilized conditions using PAb anti-Dr, followed by a secondary rhodamine isothiocyanate-labeled antibody. The intracellular markers were then labeled with appropriate antibodies under permeabilized conditions. Under these conditions, extracellular bacteria were green (GFP-labeled bacteria) and decorated with red (Dr adhesin labeling), whereas intracellular bacteria were entirely green (GFP-labeled bacteria). Cellular markers were blue.

Specimens were mounted using Dako fluorescent mounting medium (DakoCytomation SA, Trappes, France) and examined using a confocal laser scanning microscope (Zeiss LSM 510 equipped with an air-cooled argon ion laser [488 nm] and a 543-nm and 633-nm helium neon laser; Carl Zeiss, Le Pecq, France) configured with an Axiovert 100 M microscope using a Plan-Apochromat 63x/1.40 oil objective. Photographic images were resized and organized using Adobe Photoshop software (San Jose, CA).

Western blot analysis. Cells were washed once with cold PBS and then dissolved in the appropriate volume of protein-denaturing buffer and held at 100°C for 5 min. Proteins were immediately separated on 10% sodium dodecyl sulfate-polyacrylamide gels for integrin or DAF analysis and on 7% sodium dodecyl sulfate-polyacrylamide gels for CEACAM analysis. Proteins were transferred to a polyvinylidene difluoride membrane (PerkinElmer, Les Ullis, France) and examined using the Immobilon Western detection system under conditions recommended by the manufacturer (Millipore, Molsheim, France). The membranes were incubated with primary mouse anti-integrin, anti-DAF, and anti-CEACAM antibodies and then incubated with horseradish peroxidase-conjugated anti-mouse antibody (Jackson ImmunoResearch Laboratories) as the secondary antibody.

Statistical analysis. All experiments were repeated at least three times. Results were expressed as means ± the standard deviations (SD). Global F and Student's t tests were used, and a P value of <0.01 was considered significant.


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RESULTS
 
Cell association with and internalization within CHO cells of Afa/Dr DAEC does not involve {alpha}5β1 integrin. In order to find out whether the Afa/Dr adhesin and/or invasin subunits have a role in the internalization of Afa/Dr DAEC, experiments were monitored using clone B2 of CHO cells, which is deficient for {alpha}5β1 integrin expression (65). Cells were transfected with specific cDNAs encoding the human {alpha}5 (CHO-{alpha}5) or β1 (CHO-β1) integrin subunit or {alpha}5β1 (CHO-{alpha}5β1) integrin (Fig. 1A). As a control of functionality, CHO-{alpha}5β1 cells showed a positive adhesion onto fibronectin and no adhesion onto collagen (Fig. 1A). Untransfected and transfected cell lines were infected with the nonpathogenic E. coli strain AAEC185 or recombinant AAEC185DrD+E+ or AAEC185Afa-IIID+E+ bacteria, and the levels of cell-associated and internalized bacteria were determined. In infected CHO cells, a low level of bacterial cell association (4.5 log CFU/ml) was observed, which corresponded to the background level (Fig. 1B). Bacterial cell association was no greater in recombinant E. coli AAEC185DrD+E+-infected or AAEC185Afa-IIID+E+-infected CHO-{alpha}5β1 cells, regardless of whether or not the bacterium expressed Dr or Afa-III fimbriae (4.0 to 4.8 log CFU/ml), than in cells infected with the AAEC185 bacterium (Fig. 1B). Similar results were obtained with CHO-{alpha}5- and CHO-β1-infected cells (data not shown).


Figure 1
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  		FIG. 1. No cell association and internalization of Dr or AfaE-III fimbria-positive E. coli within cells engineered for expression of {alpha}5β1 integrin. Cells were infected with recombinant E. coli strains (108 CFU/ml) for 3 h at 37°C in 5% CO2/95% air. Cell-associated and internalized bacteria were determined as described in Materials and Methods. (A) Western blot analysis using MAb anti-β1 integrin subunit (clone MAR4) of total cell extracts of untransfected CHO and transfected CHO-{alpha}5β1 cells showing the absence of β1 integrin in the CHO cells and its presence in the transfected cells. PAb anti-actin was used to demonstrate equal gel loadings (left micrographs). Transfection of CHO for {alpha}5β1 integrin expression renders the cells adhering onto fibronectin but not onto collagen (right micrographs). (B) Cell-associated and internalized bacteria in CHO and CHO-{alpha}5β1 cells infected with strain AAEC185, AAEC185DrD+E+, or AAEC185Afa-IIID+E+. (C) HeLa cells were untransfected or transfected with INTERFERin alone or with two siRNAs targeting β1 integrin (β1-1 or β1-2). Western immunoblot analysis using the MAb anti-β1 integrin subunit (clone MAR4) shows the presence of β1 integrin in untransfected cells and INTERFERin-transfected cells; no β1 integrin was found in transfected siRNAs β1-1 or β1-2 cells. PAb anti-tubulin was used to demonstrate equal gel loadings. Untransfected and transfected cells were infected with AAEC185DrD+E+ bacteria. The levels of cell-associated and internalized bacteria (CFU/5 x 105 cells) were calculated as described in Materials and Methods. Each value shown is the mean ± SD from at least three independent experiments carried out in triplicate. Statistical analysis was conducted with a global F test. *, P < 0.01.

The levels of internalized bacteria were measured in untransfected and transfected cells infected with AAEC185, AAEC185DrD+E+, or AAEC185Afa-IIID+E+ bacteria. A low level of internalized bacteria was observed in CHO cells infected with AAEC185 bacteria (3.8 log CFU/ml) (Fig. 1B). No increase in internalized bacteria was observed in CHO-{alpha}5β1 infected with AAEC185DrD+E+ or AAEC185Afa-IIID+E bacteria (2.0 to 2.7 log CFU/ml) compared to those infected with AAEC185 bacteria (Fig. 1B). Similar results were observed in infected CHO-{alpha}5 and CHO-β1 cells (not shown).

In order to confirm that {alpha}5β1 integrin plays no role in the internalization of Afa/Dr DAEC, we chose to knock down β1 integrin in the HeLa cells constitutively expressing {alpha}5β1 integrin (26, 61). For this purpose, we used two siRNAs (β1-1 and β1-2). Compared with untransfected cells, no β1 integrin was detectable in either of the siRNA β1 integrin-transfected cells (Fig. 1C). As a control, we showed that β1 integrin was detectable in the HeLa cells exposed to the transfecting agent INTERFERin alone. Similar levels of cell-associated and internalized bacteria were found when the siRNA β1-1-, siRNA β1-2-, or INTERFERin-transfected cells were infected with AAEC185DrD+E+ bacteria; these did not differ from those observed in untransfected cells (Fig. 1C).

Collectively, these results demonstrate that {alpha}5β1 integrin does not function as a receptor for either the cell association or internalization of E. coli expressing Dr or Afa-III fimbriae.

Expression of DAF, CEACAM1-4L, CEA, or CEACAM6 is sufficient to induce receptor-mediated association with and internalization of DrD+E+-positive bacteria within CHO cells. We investigated whether recognition of the other epithelial receptors of Afa/Dr fimbriae, i.e., DAF, CEACAM1, CEA, or CEACAM6 (4), was sufficient to promote the internalization of Afa/Dr fimbria-positive bacteria. For this purpose, CHO cells were engineered by transfection with the specific human cDNA for the permanent expression of DAF (CHO-DAF), CEACAM1-4L (CHO-CEACAM1), CEA (CHO-CEA), or CEACAM6 (CHO-CEACAM6) (Fig. 2A). As shown in Fig. 2B, a low level of bacterial cell association (4.2 to 4.9 log CFU/ml) was observed in the untransfected CHO cells infected with WT-IH11128, AAEC185, or AAEC185DrD+E+ bacteria. When the CHO-DAF, CHO-CEACAM1, CHO-CEA, or CHO-CEACAM6 cell line was infected with the AAEC185 bacteria, low levels of cell-associated bacteria (4.0 to 5.0 log CFU/ml) were observed for each of the transfected cell lines (Fig. 2B). When infected with WT-IH11128 or AAEC185DrD+E+ bacteria, all the transfected cell lines showed similar high levels of cell-associated bacteria (7.0 to 7.7 log CFU/ml) (Fig. 2B).


Figure 2
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  		FIG. 2. Cell association and internalization of DrD+E+ fimbria-positive E. coli within CHO cells engineered to express DAF, CEACAM1, CEA, or CEACAM6 alone and to coexpress {alpha}5 or β1 integrin subunits. Cells were infected with AAEC185, WT-IH11128, or AAEC185DrD+E+ bacteria (108 CFU/ml) for 3 h at 37°C in 5% CO2/95% air. For WT-IH11128, the infection assay was conducted in the presence of 1% D-mannose to prevent type 1-pilus-mediated binding. Cell-associated and internalized bacteria were determined as described in Materials and Methods. (A) Western blot analysis using MAb D14HD11 anti-CEACAMs or MAb 8D11 anti-DAF of total cell extracts of CHO, CHO-DAF, CHO-CEACAM1, CHO-CEA, or CHO-CEACAM6 cells showing the absence of DAF and CEACAMs in CHO cells and the presence of DAF in CHO-DAF cells, CEACAM1 in CHO-CEACAM1, CEA in CHO-CEA, and CEACAM6 in CHO-CEACAM6-transfected cells. PAb anti-actin was used to demonstrate equal gel loadings. (B) Cell-associated and internalized bacteria in CHO, CHO-DAF, CHO-CEACAM1, CHO-CEA, or CHO-CEACAM6 cells infected with AAEC185, WT-IH11128, or recombinant AAEC185DrD+E+ strains. (C) Cell-associated and internalized bacteria in CHO-CEACAM1, CHO-CEACAM1 Y493F, CEACAM1 Y520F, and CEACAM1 {Delta}CT cells infected with the recombinant AAEC185DrD+E+ strain. (D) Cell-associated and internalized bacteria in CHO, CHO-DAF, CHO-CEACAM1, CHO-CEA, or CHO-CEACAM6 cells cotransfected with {alpha}5 or β1 integrin subunits and infected with the recombinant AAEC185DrD+E+ strain. In these cells, the expression of the {alpha}5 or β1 integrin subunit has been controlled by Western blot analysis (not shown). In addition, when the {alpha}5 or β1 integrin subunit was expressed in CHO cells transfected with DAF or the CEACAMs, no change in the distribution of DAF or the CEACAMs was observed (not shown). The levels of cell-associated and internalized bacteria (CFU/5 x 105 DAF- or CEACAM-positive cells) were calculated as described in Materials and Methods. Each value shown is the mean ± SD from at least three independent experiments performed in triplicate. In panel B, statistical analysis was conducted with a global F test. P was <0.01 with WT-IH11128- or AAEC185DrD+E+-infected CHO-DAF, CHO-CEACAM1, CHO-CEA, and CHO-CEACAM6 cells compared with AAEC185-infected cells.

Low levels of internalized bacteria corresponding to the background level (3.5 to 4.0 log CFU/ml) were observed in untransfected CHO cells infected with AAEC185, WT-IH11128, or AAEC185DrD+E+ bacteria (Fig. 2B). Similarly, low levels of internalized bacteria were observed when the transfected CHO-DAF, CHO-CEACAM1, CHO-CEA, or CHO-CEACAM6 cell line was infected with AAEC185 bacteria (Fig. 2B). In contrast, the transfected cell lines infected with WT-IH11128 or AAEC185DrD+E+ bacteria showed similar high levels of internalized bacteria (5.0 to 6.0 log CFU/ml) (Fig. 2B).

We next investigated the molecular requirements for CEACAM1-mediated internalization of AAEC185DrD+E+ bacteria. We observed that the phosphorylation of CEACAM1 at the cytoplasmic domain is not necessary for internalization of bacteria. Indeed, the transfected CHO cells expressing the protein mutated in Tyr493 or Tyr520 in which Dr fimbria-induced Tyr phosphorylation develops (61) showed the same levels of bacterial internalization as the cells expressing the full-length protein (Fig. 2C). In addition, we found that the cytoplasmic domain of CEACAM1 is not required for internalization, since CEACAM1-4L {Delta}CT lacking the entire cytoplasmic domain was as effective in bacterial internalization as the full-length protein (Fig. 2C).

The results described above showed that the simple expression of {alpha}5β1 integrin in CHO-B2 cells results in a lack of cell association or cell entry of Dr- or Afa-III-positive bacteria. Epithelial cells normally coexpressed {alpha}5β1 integrin and Afa/Dr fimbrial receptors, DAF, and CEACAMs. Considering this, we then investigated whether the induced coexpression of the {alpha}5 or β1 integrin subunit in cells expressing DAF, CEACAM1, CEA, or CEACAM6 increased the cell association and/or internalization of Dr fimbria-positive bacteria (Fig. 2D). For this purpose, CHO-DAF, CHO-CEACAM1, CHO-CEA, and CHO-CEACAM6 cells were cotransfected with the appropriate cDNA for the stable expression of the human {alpha}5 or β1 integrin subunit. The results showed that there was no change in either the cell association or internalization of AAEC185DrD+E+ bacteria in the infected, cotransfected cell lines expressing the {alpha}5 or β1 integrin subunit compared to that in the non-{alpha}5- or non-β1-expressing CHO-DAF, CHO-CEACAM1, CHO-CEA, and CHO-CEACAM6 cell lines (Fig. 2D).

Taken together, these findings demonstrated that the membrane-bound receptor DAF, CEACAM1, CEA, or CEACAM6 is sufficient to promote the internalization of Dr fimbria-positive bacteria. Moreover, the findings also demonstrate that the {alpha}5 or β1 integrin subunit does not act synergistically in increasing the DAF-, CEACAM1-, CEA-, or CEACAM6-dependent internalization of Dr fimbria-positive bacteria.

Microtubules and lipid rafts, but not F-actin, play a role in the internalization of DrD+E+-positive bacteria within CHO cells expressing DAF, CEACAM1, CEA, or CEACAM6. It has been previously reported that the entry of Afa/Dr DAEC into epithelial cells involves microtubules and lipid rafts but not F-actin microfilaments (26, 37, 66). We then investigated the role of F-actin microfilaments, microtubules, and lipid rafts in the internalization of AAEC185DrD+E+ bacteria into CHO-DAF, CHO-CEACAM1, CHO-CEA, and CHO-CEACAM6 cell lines. For this purpose, cells were treated with the actin-disrupting agent cytochalasin D before being infected. No change in bacterial internalization (5.7 to 6.2 log CFU/ml) was observed compared to that in untreated, infected cells (5.8 to 6.4 log CFU/ml) (Fig. 3A). The microtubule-disrupting agent nocodazole dramatically decreased the penetration of the bacteria into CHO-DAF, CHO-CEACAM1, CHO-CEA, or CHO-CEACAM6 cells (4.0 to 5.1 log CFU/ml) compared to that with untreated, infected cells (5.8 to 6.4 log CFU/ml) (Fig. 3A). After treating the cells with methyl-β-cyclodextrin, which extracts membrane-associated cholesterol and disrupts lipid rafts, the entry of AAEC185DrD+E+ bacteria into the transfected cells was dramatically lower (4.6 to 5.0 log CFU/ml) than that into untreated, infected cells (5.8 to 6.4 log CFU/ml) (Fig. 3A). When the cells were treated with nocodazole plus methyl-β-cyclodextrin, there was an insignificant change in bacterial internalization compared to cells treated with nocodazole alone or methyl-β-cyclodextrin alone (Fig. 3). For the drugs modifying bacterial cell entry, it was noted that no change in the cell association of AAEC185DrD+E+ bacteria was observed after drug treatments (Fig. 3B).


Figure 3
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  		FIG. 3. Microtubules and lipid rafts but not F-actin microfilaments were involved in internalization of AAEC185DrD+E+ bacteria within CHO-DAF, CHO-CEACAM1, CHO-CEA, and CHO-CEACAM6 cells. Cells were infected with E. coli (108 CFU/ml) for 3 h at 37°C in 5% CO2/95% air. Treatments with disrupting agents are described in Materials and Methods. The levels of cell-associated (B) and internalized (A) bacteria (CFU/5 x 105 DAF- or CEACAM-positive cells) were calculated as described in Materials and Methods. Each value shown is the mean ± SD from at least three independent experiments performed in triplicate. In panels A and B, statistical analysis was conducted with a global F test. *, P < 0.01 for treated cells versus untreated cells.

DraE or AfaE-III adhesin subunit is sufficient to promote bacterial internalization. In order to examine whether the DraE or AfaE-III adhesin subunit is necessary to induce internalization of Afa/Dr DAEC, we used CHO cells transfected for coexpression of DAF and {alpha}5β1 integrin. A nonpolar mutation that deletes the entire draD gene was monitored in the WT-IH11128 strain (WT-IH11128D–E+), and site-directed mutagenesis was conducted in the draD gene in order to produce strain AAEC185DrD–E+. We also used recombinant AAEC185 strains expressing Afa-III fimbriae (AAEC185Afa-IIID+E+), Afa-III fimbriae in which the afaD gene had been deleted (AAEC185Afa-IIID–E+), Afa-III fimbriae in which the afaE gene had been deleted (AAEC185Afa-IIID+E–), or Afa-III fimbriae in which both the afaE and afaD genes had been deleted (AAEC185Afa-IIID–E–) (20, 35). Low levels of cell-associated and internalized bacteria, similar to those of the background level, were observed in CHO-DAF-{alpha}5β1 cells infected with AAEC185 bacteria (Fig. 4). In contrast, similarly high levels of cell-associated and internalized bacteria were observed in cells infected with WT-IH11128, AAEC185DrD+E+, or AAEC185Afa-IIID+E+ (Fig. 4). Interestingly, when these cells were infected with the three mutated bacterial strains, i.e., WT-IH11128D–E+, AAEC185DrD–E+, and AAEC185Afa-IIID–E+, the levels of cell-associated or internalized bacteria were no lower than those in the cells infected with WT-IH11128, AAEC185DrD+E+, and AAEC185Afa-IIID+E+ (Fig. 4). In contrast, when the cells were infected with the two mutated bacterial strains, i.e., AAEC185Afa-IIID+E– and AAEC185Afa-IIID–E–, there was a dramatic decrease in the levels of cell-associated or internalized bacteria compared with those infected with WT-IH11128, AAEC185DrD+E+, AAEC185Afa-IIID+E+, WT-IH11128D–E+, AAEC185DrD–E+, or AAEC185Afa-IIID–E+ (Fig. 4). Similar results were obtained with HeLa cells constitutively expressing DAF (24, 27, 35) and {alpha}5β1 integrin (26, 61) and with CHO-DAF-{alpha}5β1 cells (not shown). Taken together, these results show that DraE and AfaE-III adhesin subunits are sufficient to permit the bacterial internalization of Afa/Dr DAEC into epithelial cells.


Figure 4
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  		FIG. 4. DraE and AfaE-III adhesin subunits are sufficient to promote the internalization of WT-IH11128, AAEC185Dr, and AAEC185Afa-III bacteria within CHO-DAF-{alpha}5β1 cells. CHO-DAF-{alpha}5β1 cells were infected with WT-IH11128D+E+, WT-IH11128D–E+, AAEC185DrD+E+, AAEC185DrD–E+, AAEC185Afa-IIID+E+, AAEC185Afa-IIID–E+, AAEC185Afa-IIID+E–, or AAEC185Afa-IIID–E– bacteria (108 CFU/ml) for 3 h at 37°C in 5% CO2/95% air. For WT-IH11128D+E+ and WT-IH11128D–E+, the infection assay was conducted in the presence of 1% D-mannose to prevent type 1-pilus-mediated binding. Cell-associated and internalized bacteria were determined as described in Materials and Methods. The levels of cell-associated and internalized bacteria (CFU/5 x 105 DAF-positive cells) were calculated as described in Materials and Methods. Each value shown is the mean ± SD from at least three independent experiments performed in triplicate. *, P < 0.01 versus AAEC185.

DraE or AfaE-III adhesin subunits are sufficient to promote {alpha}5β1 integrin clustering. It has been previously reported that clustering of {alpha}5β1 integrin develops around cell-adhering Dr fimbria-positive E. coli (37), Afa-III-positive bacteria (61), and Afa-III-coated beads (14). As shown in Fig. 5, WT-IH11128, AAEC185DrD+E+, and AAEC185Afa-IIIE+D+ bacteria adhering to CHO-DAF-{alpha}5β1 cells are decorated by β1 integrin-positive immunolabeling. It was noted that the randomly distributed nonadhering AAEC185 bacteria showed an absence of β1 integrin-positive immunolabeling (not shown). We next investigated whether mutations in the D or E adhesin subunit affect the capacity of bacteria to mobilize the β1 integrin. As shown in Fig. 5, WT-IH11128D–E+, AAEC185DrD–E+, and AAEC185Afa-IIID–E+ mutated bacteria have conserved their abilities to mobilize β1 integrin. Moreover, E-negative bacteria are nonadhering, and the sparsely distributed bacteria showed no recruitment of β1 integrin (not shown). These results indicate that the DraE or AfaE-III adhesin subunit is sufficient to induce the mobilization of β1 integrin around adhering bacteria.


Figure 5
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  		FIG. 5. DraE and AfaE-III adhesin subunits are sufficient to promote the clustering of β1 integrin subunits around adhering bacteria. CHO-DAF-{alpha}5β1 cells were infected with WT-IH11128D+E+, WT-IH11128D–E+, AAEC185DrD+E+, AAEC185DrD–E+, AAEC185Afa-IIID+E+, or AAEC185Afa-IIID–E+ bacteria (108 CFU/ml) for 3 h at 37°C in 5% CO2/95% air. For WT-IH11128D+E+ and WT-IH11128D–E+, the infection assay was conducted in the presence of 1% D-mannose to prevent type 1-pilus-mediated binding. Nonpermeabilized cells were fixed and coimmunolabeled as described in Materials and Methods to detect β1 integrin (red) using MAb anti-β1 integrin (clone P5D2) and adhering bacteria using PAb anti-Dr adhesin (green). Arrows indicate the bacteria decorated by positive β1 integrin immunolabeling. Merged images show the colocalization of β1 integrin and adhering bacteria. Micrographs are representative of three experiments.

Intracellularly localized WT-IH11128 bacteria survived for a long time. We investigated how long the WT-IH11128 bacteria survived after internalization within CHO-DAF, CHO-CEACAM1, CHO-CEA, and CHO-CEACAM6 cells. We determined the levels of internalized, viable WT-IH11128 bacteria as a function of the time postinfection (Fig. 6A). For the three cell lines expressing the different CEACAMs, the number of surviving bacteria declined steadily, and only 0.5 to 2.0 log CFU/ml were found intracellularly at 96 h postinfection. In comparison, the number of viable intracellular WT-IH11128 bacteria was higher in CHO-DAF cells, and the level of viable intracellular WT-IH11128 bacteria in CHO-DAF cells stabilized 48 h postinfection (3 log CFU/ml) and remained stable until 96 h postinfection.


Figure 6
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  		FIG. 6. Intracellular survival of WT-IH11128 and AAEC185DrD+E+ bacteria in CHO-DAF, -CEACAM1, -CEA, -CEACAM6, and HeLa cells. Cells were infected (108 CFU/ml) for 3 h at 37°C in 5% CO2/95% air. For WT-IH11128D+E+ and WT-IH11128D–E+, the infection assay was conducted in the presence of 1% D-mannose to prevent type 1-pilus-mediated binding. The infected cells were then incubated for 1 h in a medium containing 100 µg/ml of gentamicin to kill the extracellular adhering bacteria. The infected cells were subsequently cultured in the presence of cell culture medium containing gentamicin (15 µg/ml) to prevent replication of any remaining viable extracellular bacteria. Internalized bacteria were determined 24, 48, 72, and 96 h postinfection as described in Materials and Methods. The levels of internalized bacteria (CFU/5 x 105 DAF- or CEACAM-positive cells) were calculated as described in Materials and Methods. Each value shown is the mean ± SD from at least three independent experiments performed in triplicate. (A) Intracellular survival of WT-IH11128 bacteria in CHO-DAF, -CEACAM1, -CEA, and -CEACAM6 cells. At 96 h postinfection, Student's t test determined a P value of <0.01 for CHO-CEACAM1 and CHO-CEACAM6 cells and a P value of <0.05 for CHO-CEA cells versus CHO-DAF cells. (B) Comparison of rates of intracellular survival of WT-IH11128 and AAEC185DrD+E+ bacteria in HeLa cells. Student's t test determined a P value of <0.01 (72 h postinfection) for AAEC185DrD+E+ and a P value of <0.05 (96 h postinfection) for WT-IH11128.

We next compared the rates of survival of WT-IH11128 bacteria and recombinant Dr fimbria-expressing bacteria (AAEC185DrD+E+) within HeLa cells. As shown in Fig. 6B, HeLa cells infected with the WT-IH1128 bacteria showed that the time course of the survival of intracellular bacteria was similar to that observed in WT-IH11128-infected CHO-DAF cells (Fig. 6A), indicating that the phenomenon is not cell line dependent. In HeLa cells infected with AAEC185DrD+E+ bacteria, the survival of intracellular bacteria was significantly lower than that in HeLa cells infected with WT-IH11128 bacteria. These findings suggest that a bacterial factor(s) other than Dr fimbriae is required for the intracellular survival of WT-IH11128 bacteria.

Intracellularly localized WT-IH11128 bacteria reside in a vacuole expressing endosomal/lysosomal markers. Scanning electron microscopy studies have previously shown that internalized Afa/Dr DAEC cells expressing Dr (15, 66) or Afa-III (61) fimbriae resided within large vacuoles. Considering that the vacuole membrane-associated endosomal/lysosomal markers of Afa/Dr DAEC-containing vacuoles have not been identified, we conducted such an identification in WT-IH11128-infected HeLa cells 24 h postinfection. We found that the internalized WT-IH11128 bacteria were located in vacuoles containing more than one bacterium (Fig. 7). Since WT-IH11128 bacteria bind to DAF endogenously expressed at the cell surfaces of HeLa cells, we tried to find out whether DAF was associated with the WT-IH11128-containing vacuoles. As shown in Fig. 7, no positive DAF immunolabeling was observed at the membranes of the WT-IH11128-containing vacuoles. Experiments were performed to investigate the acquisition of endosomal/lysosomal markers by WT-IH11128-containing vacuoles using antibodies directed against late endosomal markers, Lamp-1 and Lamp-2, CD63, and cathepsin D. Positive immunolabeling of Lamp-1, Lamp-2, and CD63 was observed in the WT-IH11128-containing vacuoles (Fig. 7). In contrast, no labeling of the luminal hydrolase, cathepsin D, was observed in the WT-IH11128-containing vacuoles (Fig. 7). In addition, the WT-IH11128-containing vacuoles displayed the presence of LysoTracker, indicating that they are acidic (Fig. 7). It was noted that the AAEC185DrD+E+-containing vacuoles expressed the same markers as those found in the WT-IH11128-containing vacuoles (not shown).


Figure 7
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  		FIG. 7. Localization of WT-IH11128 bacteria within infected HeLa cells 24 h postinfection shows WT-IH11128-containing vacuoles positive for Lamp-1, Lamp-2, CD63, and LysoTracker but not for DAF or cathepsin D. Cells were infected with GFP-WT-IH11128 bacteria (108 CFU/ml) for 3 h at 37°C in 5% CO2/95% air in the presence of 1% D-mannose to prevent type 1-pilus-mediated binding. The infected cells were treated and subcultured with gentamicin to prevent extracellular bacterial growth as described in the legend to Fig. 6. Cells were fixed and coimmunolabeled as described in Materials and Methods to detect markers, intracellular and extracellular bacteria, DAF, Lamp-1, Lamp-2, CD63, cathepsin D, or LysoTracker (blue). Intracellular bacteria are green labeled, and extracellular bacteria are green and red labeled. Merged images show the colocalization of Lamp-1, Lamp-2, and CD63 (blue) and intracellularly localized bacteria (green). No colocalization was seen of DAF and cathepsin D (blue) and intracellularly localized bacteria (green). For LysoTracker, a merged close-up image shows the colocalization of LysoTracker fluorescence (blue) and intracellularly localized bacteria (green). Arrows indicate the colocalization or lack of colocalization of intracellular bacteria and markers. Micrographs are representative of at least three experiments.

Cellular autophagy is a process that leads to degradation of the cytosolic structure, and some bacteria are vulnerable to autophagic destruction, whereas others have developed successful avoidance strategies (39). We investigated the possible involvement of the autophagic pathway in the intracellular lifestyle of WT-IH11128 bacteria. For this purpose, we used HeLa cells transfected with the plasmid encoding an autophagosomal marker (2), the GFP-LC3 protein. When cells were infected with WT-IH11128 bacteria, no GFP-LC3 was found with the WT-IH11128-containing vacuoles (Fig. 8A). In order to confirm that autophagy does not play any role in the intracellular lifestyle of WT-IH11128 bacteria, HeLa cells were treated with rapamycin, a known inducer of autophagy (13). As expected, the level of punctate GFP-LC3 was higher in the rapamycin-treated cells than in untreated cells (Fig. 8B). Interestingly, when the rapamycin-treated cells were infected with WT-IH11128 bacteria, the time course of survival of the internalized WT-IH11128 bacteria was the same as that in untreated, infected cells (Fig. 8C).


Figure 8
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  		FIG. 8. WT-IH11128-containing vacuoles do not interact with the autophagic pathway. HeLa-GFP-LC3 cells stably transfected for the expression of the GFP-labeled autophagosomal marker, GFP-LC3, were infected with WT-IH11128 bacteria (108 CFU/ml) for 3 h at 37°C in 5% CO2/95% air in the presence of 1% D-mannose to prevent type 1-pilus-mediated binding. The infected cells were treated and subcultured with gentamicin to prevent extracellular bacterial growth as described in the legend to Fig. 6. (A) Cells were fixed and immunolabeled as described in Materials and Methods for the detection of intracellular bacteria (green) and extracellular bacteria (green and red). Merged close-up images show that there was no colocalization of GFP-LC3 (blue) and intracellular (green) bacteria. Micrographs are representative of three experiments. (B) HeLa-GFP-LC3 cells were treated with rapamycin (500 nM) to activate autophagy. Micrographs show that punctate GFP-LC3 was greater in rapamycin-treated cells than in untreated cells. (C) The graph shows that the intracellular survival of WT-IH11128 bacteria was the same in rapamycin-treated cells as in untreated cells. The levels of internalized bacteria (CFU/5 x 105 DAF-positive cells) were calculated as described in Materials and Methods. Each value shown is the mean ± SD from at least three independent experiments performed in triplicate.


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DISCUSSION
 
In this work, we used transfected epithelial cells as a model to investigate the mechanism mediating the Afa/Dr adhesin subunit- and/or invasin subunit-dependent internalization and the intracellular lifestyle of Afa/Dr DAEC. For this purpose, we transfected CHO cells with all the known epithelial cell receptors of Afa/Dr adhesin subunits, i.e., DAF, CEACAM1, CEA, or CEACAM6, or with the receptor of Afa/Dr invasin subunits, i.e., the β1 integrin subunit or {alpha}5β1 integrin. We provide evidence that neither the {alpha}5 or β1 integrin subunit nor {alpha}5β1 integrin functioned as a receptor mediating the adhesion and/or internalization of Dr or Afa-III fimbria-positive bacteria. Moreover, we found that the induced coexpression of the {alpha}5 or β1 integrin subunit in CHO cells expressing DAF, CEACAM1-4L, CEA, or CEACAM6 did not increase the internalization of Dr fimbria-positive bacteria, indicating that integrin does not act as a coreceptor during the internalization of Afa/Dr DAEC. Finally, by silencing the gene encoding the β1 integrin subunit using a specific siRNA, we demonstrated that the β1 integrin subunit plays no role in the internalization of Dr fimbria-positive bacteria into epithelial cells. These findings are consistent with those of Cota et al. (14), showing that (i) beads coated with AfaD-III protein did not associate and did not internalize into HeLa cells and that (ii) beads coated with AfaED-III proteins interacted with HeLa cells and were internalized as a result of the fact that the AfaE-III adhesin subunit recognized the DAF constitutively expressed by HeLa cells. According to those authors, the fact that beads coated with the AfaD-III protein were not internalized was attributable to the low affinity of the AfaD-III protein for {alpha}5β1 integrin, which contrasted with the situation during a Yersinia invasion (34). In contrast, the results reported here differed markedly from previous reports, including some from our group. Indeed, Guignot et al. (26) have reported that the entry of Dr fimbria-positive E. coli cells into polarized intestinal epithelial Caco-2 cells occurs specifically via the basolateral domain of the cells and that bacterial internalization is blocked by an anti-{alpha}5β1 integrin antibody. Plançon et al. (61) have shown that AfaD-III-coated beads penetrated into undifferentiated Caco-2 and HeLa epithelial cells and into F9-TKO cells, regardless of whether they expressed the β1 integrin subunit, and that this was inhibited by a monoclonal antibody directed against the β1 integrin subunit. The discrepancy between these previous reports and the present data may be attributable to the fact that, particularly as a result of the nonspecific occupancy of a membrane-bound Dr fimbrial receptor(s) by the antibody, the anti-integrin antibody may be less specific than the strategies used here, including the specific cell expression obtained by the transfection of cDNAs coding for integrin subunits or gene silencing. It is interesting to note that our results regarding the role of Afa/Dr DAEC in UTIs revealed that the internalization mechanism of these bacteria differed from that used by UPEC cells expressing type 1 pili, for which the {alpha}3 and β1 integrins are functional receptors for FimH-induced cell association and internalization (18).

We demonstrated that whether or not the AfaD or DraD invasin subunits were present, there was no difference in the cell association and internalization of bacteria expressing DraE adhesin subunit in cells expressing the {alpha}5 or β1 integrin subunit or {alpha}5β1 integrin. The question that remains is why do Afa/Dr DAEC strains by the AfaE/DraE adhesin subunit trigger the bacterial mobilization of {alpha}5β1 integrin. One interesting hypothesis is that independently of bacterial internalization, Afa/Dr DAEC could induce lipid raft- and {alpha}5β1 integrin-dependent signaling. Indeed, under certain conditions, including clustering (42), integrins associate with cholesterol-rich membrane microdomains (32). Association of β3 integrin with CEACAM1, which depends on the phosphorylation of Tyr-488 in the CEACAM1 cytoplasmic domain, has already been reported (9). Ordonez et al. (59) have recently demonstrated that {alpha}5β1 integrin represents a nodal element in a multitude of biological effects mediated by CEA/CEACAM6 that imply coclustering of the molecules (10). Moreover, it has been proposed that interaction with {alpha}5β1 integrin accounts for the tumorigenic effects of CEA/CEACAM6 (28, 33, 58) and DAF (49). In the future, it could be interesting to find out whether the AfaE, DraE, or DaaE adhesin subunit-induced mobilization of {alpha}5β1 integrin within lipid rafts together with DAF or CEACAMs triggers integrin-dependent cell signaling and, if so, which cell response(s) is triggered downstream.

Using a recombinant strain expressing Dr fimbriae in which a stop codon has been inserted into draD and a recombinant strain expressing Afa-III fimbriae in which the afaD gene has been deleted, we found that DraE or AfaE-III adhesin subunits are sufficient to promote the entry of bacterial cells into the transfected CHO-DAF cells and HeLa cells constitutively expressing DAF. These results are in agreement with previous reports by Nowicki and coworkers (15, 24, 66) showing that the DraE adhesin subunit is necessary and sufficient to trigger the DAF receptor-mediated internalization of WT-IH11128 bacteria. We provide new insight showing that, like that of DAF, recognition by the DraE and AfaE-III adhesin subunits of CEACAM1, CEA, and CEACAM6 is sufficient to induce the internalization of Dr or Afa-III fimbria-positive bacteria. Taken together, these findings demonstrate that the DraE and AfaE-III adhesin subunits are sufficient to promote the receptor-mediated internalization of Afa/Dr DAEC into epithelial cells expressing the receptors of Afa/DrDAF or Afa/DrCEACAM fimbriae.

The molecular requirements for DAF-mediated internalization of Dr fimbria-positive E. coli within epithelial cells have been previously examined, showing the pivotal role of the extracellular CCP3 domain and the glycosylphosphatidylinositol (GPI) anchor (66). For the recognition of CEACAMs by Afa/Dr fimbria-positive E. coli, Korotkova et al. (40) have demonstrated the pivotal role of the N-terminal domain of the proteins as it occurs for neisserial opacity-associated (Opa) proteins (71). The present results revealed that the internalization process of Afa/Dr fimbria-positive bacteria after recognizing CEACAM1, CEA, and CEACAM6 resembles in part the process developed by Neisseria gonorrhoeae and Neisseria meningitidis expressing colony Opa proteins (5, 47, 64). In particular, we found that the phosphorylation of CEACAM1 at its cytoplasmic tail was not required for the internalization of AAEC185DrD+E+ bacteria as it has been observed for CEACAM1-mediated internalization of Neisseria gonorrhoeae (51). Moreover, as found for Neisseria gonorrhoeae (51), we observed that the cytoplasmic domain of CEACAM1 was not necessary for the internalization of AAEC185DrD+E+ bacteria.

Our data with CHO-DAF cells confirmed the previously reported role of the microtubule network for the internalization of Dr fimbria-positive E. coli within epithelial cells expressing DAF (22, 26) and was the first report of the role of the microtubule network in the CEACAM1, CEA, and CEACAM6 receptor-mediated internalization of Dr fimbria-positive E. coli within epithelial cells. Microbial pathogens exploit lipid rafts on the host cell plasma membrane, using them as signaling platforms and/or entry sites into the cell (43). We found that the internalization of WT-IH11128 bacteria into CHO-DAF, -CEACAM1, -CEA, and -CEACAM6 cells was dramatically altered when the cells were treated with the lipid raft-disorganizing agent methyl-β-cyclodextrin. For DAF, these results confirm previous findings in different epithelial cell lines (26, 37, 66) and first described the role of lipid rafts in the CEACAM1, CEA, and CEACAM6 receptor-mediated internalization of Dr fimbria-positive E. coli into epithelial cells. The same lipid raft dependency has recently been observed for the CEA- or CEACAM6-mediated internalization of Opa52-expressing N. gonorrhoeae (64). It is not surprising that the internalization of Dr fimbria-positive E. coli after recognition of CEA and CEACAM6 is lipid raft dependent in view of the GPI anchor of these proteins and the well-established association of GPI-anchored proteins with lipid rafts (68). The fact that the hCEACAM1-4L receptor-mediated internalization of Dr fimbria-positive E. coli can be influenced by disruption of cholesterol-rich membrane microdomains is more surprising in view of the transmembrane attachment of the molecule. Interestingly, our group has recently demonstrated that CEACAM1-4L can be translocated into lipid rafts following epithelial cell infection by Dr fimbria-positive E. coli (62). Moreover, Schmitter et al. (64) have reported that the CEACAM1-mediated internalization of Opa52-expressing N. gonorrhoeae relates to lipid rafts. Muenzner et al. (51) have observed recently that the transmembrane domain of CEACAM1-4L plays a pivotal role in the translocation of this protein into cholesterol-rich membrane microdomains and that, consistently with our results, the disorganization of lipid rafts severely blocks the CEACAM1-4L-dependent uptake of OpaCEA-expressing N. gonorrhoeae, N. meningitis, Haemophilus influenzae, and Moraxella catarrhalis.

Characterization of the endosomal/lysosomal markers acquired during maturation of the late WT-IH11128-containing vacuoles revealed that these vacuoles displayed some of the features of late endosomes, including the presence of Lamp-1, Lamp-2, and CD63 proteins but not of cathepsin D, and were acidic. We also found that the maturation of WT-IH11128-containing vacuoles in epithelial cells did not involve interaction with the autophagic pathway. The maturation of the WT-IH11128-containing vacuoles has some similarity with the previously described intracellular lifestyle of the UPEC strain UTI89, which expresses type 1 pili. Indeed, the UPEC UTI89-containing vacuoles are both Lamp-1 positive and CD63 positive in 5637 bladder epithelial cells (19), are Lamp-1 positive and cathepsin D negative in the bladder of C57BL/6 mice (54), and are acidic, accumulating the acidotropic reagent DAMP (19).

Some invasive E. coli isolates have developed sophisticated strategies to prevent normal phagosomal maturation and/or resist the intraphagosomal killing process. UTIs, including cystitis and pyelonephritis, are characterized by a tendency to recur and to develop into chronic maladies. So far, host and UPEC factors that contribute to recurrence and chronicity have been only partly characterized (8). Here, we report that internalized WT-IH11128 bacteria survived better within transfected CHO-DAF cells than bacteria internalized within CHO-CEACAM1-4L, -CEA, or -CEACAM6 cells. It was interesting to note that in cells expressing CEA or CEACAM6, the intracellular survival of WT-IH11128 bacteria appeared to be markedly longer than that of Opa57-expressing gonococci, which had been wiped out 6 h postinfection (47). Mulvey et al. (52) have observed that in 5637 bladder epithelial cells, the uropathogenic strains NU14 and UTI189 survived better than the recombinant E. coli K-12 strain AAEC185/pSH2 expressing type 1 pili. The mechanisms controlling the intracellular growth and recurrence of UPEC have particularly been investigated for UPEC cells that express type 1 pili (8, 53). Two intracellular localizations have been observed. The first consists of the type 1-pilus-dependent formation of intracellular bacterial communities (52) displaying the characteristics of a biofilm (36). Interestingly, biofilm-forming bacteria have been described that display the particular characteristic of forming small subpopulations of persister cells in a dormant state (44). The second form of UPEC intracellular localization involves the formation of vacuoles containing bacteria in pairs, quadruplets, or small clusters, known as "quiescent intravacuolar reservoirs" (36). The intracellular WT-IH11128-containing vacuole observed here seems to resemble type 1-pilus-UPEC quiescent intravacuolar reservoirs. Our results suggest that WT-IH11128 has developed sophisticated strategies to survive intracellularly, and this process could explain the recurrence of Afa/Dr DAEC-dependent UTIs (29, 30, 57).

In conclusion, the results reported here demonstrate that the recognition of DAF, CEACAM1, CEA, or CEACAM6 by the AfaE and DraE adhesin subunits is sufficient to promote the microtubule-dependent internalization of Afa/Dr DAEC into epithelial cells. In addition, our data also demonstrate that AfaD-III and DraD invasin subunits and {alpha}5β1 integrin play no role in the cell association and internalization of Afa/Dr DAEC within epithelial cells. These results are consistent with the mechanism proposed by the Nowicki group (15, 23-25, 66) in which the DraE adhesin subunit functions both as an adhesin and an invasin. In addition, the results first demonstrated that internalized Dr fimbria-positive bacteria survived intracellularly within vacuoles displaying some of the features of late endosomes.


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ADDENDUM
 
During manuscript submission, Korotkova et al. (41) reported similar results showing the DraE-mediated internalization and mobilization of β1 integrin in CHO cell transfectants expressing DAF or CEACAMs. In addition, those authors have also demonstrated that the DraD subunit, previously implicated as an "invasin," is not required for β1 integrin recruitment or bacterial internalization.


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ACKNOWLEDGMENTS
 
This study used the Plateforme d'Imagerie Cellulaire (IFR141, Châtenay-Malabry, France). We are particularly grateful to E. Craig and P. Mold (Manchester University, United Kingdom) for their generous gift of CHO-B2 cells. We also thank S. L. Moseley (School of Medicine, University of Washington, Seattle, WA) for the pCC90 plasmid encoding Dr fimbriae; C. Le Bouguenec (Unité de Pathogénie Bactérienne des Muqueuses, Institut Pasteur, Paris, France) for the pIlL1151, pILL1168, pIIL1169, and pILL570 plasmids encoding AfaE-III fimbriae and mutated fimbriae, respectively; I. Anegon (INSERM U437, Nantes, France) for the pDR2{Delta}EFDAF plasmid encoding human DAF; C. Sasakawa (Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo, Japan) for pECE plasmids encoding {alpha}5 and β1 integrins; and A. M. Tolkovsky (Cambridge University, United Kingdom) for her gift of the HeLa-GFP-LC3 cell line. We thank D. M. Lublin (Washington University School of Medicine, Saint Louis, MO) for the generous gift of the MAb anti-DAF. We thank M. Vasseur for her kindness in doing the statistical analysis.

J.G. and A.L.S. conceived and designed the experiments and analyzed the data. J.G., S.H., I.K., and I.C. performed the experiments. A.L.S. wrote the manuscript.

This work was supported by institutional French funding from the Institut National de la Santé et de la Recherche Médicale (Inserm), Université Paris-Sud 11, the Ministère de la Recherche (A.L.S.), and the Institut National de la Recherche Agronomique (Inra) (S.H.).


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FOOTNOTES
 
* Corresponding author. Mailing address: UMR756, Faculté de Pharmacie, 5 Rue J. B. Clément, 92296 Châtenay-Malabry Cedex, France. Phone: 33-1 46 83 56 61. Fax: 33-1 46 83 58 44. E-mail: alain.servin{at}u-psud.fr Back

{triangledown} Published ahead of print on 17 November 2008. Back

Editor: B. A. McCormick


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Infection and Immunity, January 2009, p. 517-531, Vol. 77, No. 1
0019-9567/09/$08.00+0     doi:10.1128/IAI.00695-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.




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