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INFECTION AND IMMUNITY, 0019-9567/99/$04.0010 July 1999, p. 3302–3307 Vol. 67, No. 7 Copyright © 1999, American Society for Microbiology. All Rights Reserved. Role of Lactosyl Glycan Sequences in Inhibiting Enteropathogenic Escherichia coli Attachment ROSA P. VANMAELE, LOUIS D. HEERZE, AND GLEN D. ARMSTRONG* Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta T6G 2H7, Canada Received 23 December 1998/Returned for modification 24 February 1999/Accepted 12 April 1999 Previously, we found that asialo-lactosamine sequences served as receptors for enteropathogenic Escherichia coli (EPEC) binding to Chinese hamster ovary (CHO) cells. In the present report, we have extended these earlier results by examining the ability of lactosamine- or fucosylated lactosamine-bovine serum albumin (BSA) glycoconjugates to inhibit EPEC, strain E2348/69, binding to HEp-2 cells. We found that, consistent with our previous findings with CHO cells, N-acetyllactosamine-BSA was the most effective inhibitor of EPEC localized adherence to HEp-2 cells, with Lewis X-BSA being the next best inhibitor. Further investigation revealed that coincubating EPEC E2348/69 with these BSA glycoconjugates alone caused a decrease in the expression of the bundle-forming pilus structural subunit (BfpA) and intimin by the bacteria. BfpA and intimin expression were reduced to the greatest extent by N-acetyllactosamine–BSA and Lewis X-BSA, respectively. These results suggest that the glycoconjugate inhibition of EPEC binding to HEp-2 cells might be achieved, wholly or in part, by an active mechanism that is distinct from simple competitive antagonism of receptor- adhesin interactions. Enteropathogenic Escherichia coli (EPEC) is a significant cause of diarrhea in young children, especially those less than 6 months of age (7, 45). Clinical symptoms are associated with intestinal attaching and effacing (A/E) lesions, which are char- acterized by the degeneration of microvilli, and the intimate association of bacteria with pedestal-like structures formed from host epithelial cells (3, 28, 34, 41). Donnenberg and Kaper originally proposed that EPEC colonization of intestinal epithelial surfaces occurs by a three-stage process (10). Ac- cording to this multistep model, the initial binding of EPEC to intestinal epithelial cells involves their nonintimate attachment as microcolonies. Next, the bacteria induce signal transduction processes in host cells, which results in cytoskeletal rearrange- ment and the effacement of surface microvilli. In the final stage of attachment, the cytoskeletal components are organized to form pedestal-like structures which partially surround adher- ent organisms. The close proximity of bacteria to these pedes- tal-like structures constitutes the intimate attachment charac- teristic of A/E lesions. More recently, Hicks et al. proposed a modified model for EPEC colonization. This model is similar to the three-stage model of Donnenberg and Kaper except that three-dimensional microcolonies are thought to develop after, not before, intimate attachment (15). Since adherence is an important factor in EPEC pathogen- esis, considerable research has been performed to identify bac- terial and eukaryotic cell structures involved in attachment. So far, two bacterial structures have been relatively well charac- terized. The first, bundle-forming pili (BFP), are associated with the attachment of EPEC as microcolonies to discrete sites on epithelial cells, a pattern which is referred to as localized adherence (LA) (4, 14, 36). Scanning electron micrographs of LA EPEC revealed that BFP are involved in mediating inter- bacterial linkages within microcolonies. Whether the BFP also function as adhesins for EPEC binding to epithelial cells re- mains to be resolved, however, since these structures appear to mediate bacterial binding to HEp-2 cells but not to human intestinal tissue in organ culture (14, 15, 40). A second bacte- rial protein involved in attachment is intimin (18, 19). This outer membrane protein is necessary for the intimate attach- ment of EPEC to epithelial cells and functions to focus host cell cytoskeletal components beneath adherent bacteria to form A/E lesions (31). In contrast to what is known about bacterial structures in- volved in attachment, EPEC receptors on eukaryotic cells are less well characterized. Rosenshine et al. originally identified a 90-kDa tyrosine-phosphorylated protein (Hp90) in epithelial cell membranes which bound intimin (33). This protein was subsequently shown to be a secreted bacterial protein, called Tir (translocated intimin receptor), which, after translocation into the eukaryotic cell, becomes phosphorylated and serves as the receptor for intimin (6, 21). Frankel et al. reported that intimin may also bind to b1 integrins (13). In addition to these reports, several groups have identified oligosaccharide structures that are potential receptors for EPEC. By using various experimental approaches, N-acetyl- galactosamine (35), fucosylated milk oligosaccharide se- quences (5), GM3 gangliosides (16), and the GalNAcb(134) Gal portion of asialo-GM1 and asialo-GM2 structures (17) have all been implicated in EPEC attachment to eukaryotic cells. As well, we previously reported that EPEC bound to asialo-lactosamine sequences of N-linked glycoproteins on Chinese hamster ovary (CHO) cells (43). A possible role for O-linked glycoproteins or glycolipids in EPEC attachment to CHO cells was also suggested by data presented in our previ- ous report. Several reports suggest that, in general, EPEC recognize lactosyl structures on epithelial cells. However, additional car- bohydrate residues (i.e., sialic acid and fucose) are frequently attached to these core structures. While our previous results with CHO cell Lec mutants indicated that EPEC do not re- quire sialic acid in order to bind, the importance of fucose in these interactions was not addressed since CHO cells do not express certain fucosylated glycans (30). In consideration of * Corresponding author. Mailing address: Medical Microbiology and Immunology, 1-21 Medical Sciences Bldg., University of Alberta, Edmonton, Alberta T6G 2H7, Canada. Phone: (780) 492-2303. Fax: (780) 492-7521. E-mail: glen.armstrong@ualberta.ca. 3302 previous results which suggested that fucosylated structures are preferentially recognized by EPEC (5), we performed bind- ing inhibition experiments with synthetic lactosamine or fuco- sylated lactosamine bovine serum albumin (BSA)-glycoconju- gates to investigate the role of fucose in EPEC attachment to epithelial cells. Our results demonstrated that N-acetyllac- tosamine (LacNAc)-BSA, followed by Lewis X (LeX)-BSA, were the most effective soluble inhibitors of EPEC E2348/69 attachment to HEp-2 cells. Furthermore, the interaction of bacteria with these specific glycoconjugates, alone, caused a decrease in the expression of BfpA, the structural subunit of BFP (9, 38), and intimin, in these organisms. These results suggest that EPEC may possess a mechanism for regulating the expression of these proteins which could contribute to the reduced ability of the bacteria to bind to HEp-2 cells. MATERIALS AND METHODS Reagents. All glycoconjugates consisted of chemically synthesized oligosaccha- ride sequences (Table 1) conjugated to BSA through an 8-methoxycarbonyloctyl linker arm (25). LacNAc-BSA and Lewis Y (LeY)-BSA were generously pro- vided by O. Hindsgaul (University of Alberta, Edmonton, Alberta, Canada). LeX-BSA was purchased from the Alberta Research Council (Edmonton, Al- berta, Canada). The incorporation of ligands into BSA was determined by mass spectroscopy to be as follows (in mol/mol): LacNAc-BSA, 19:1; LeX-BSA, 26:1; and LeY-BSA, 17:1. All glycoconjugates were solubilized in phosphate-buffered saline (PBS) (5 mg/ml) and stored at 220°C prior to use. Polyclonal rabbit anti-intimin (18) and anti-BfpA (47) antibodies were kindly provided by J. B. Kaper and M. S. Donnenberg (University of Maryland School of Medicine, Baltimore), respectively. Rabbit anti-maltose-binding protein (anti-MBP) anti- bodies were purchased from New England Biolabs (Mississauga, Ontario, Can- ada). Bacterial strains. EPEC E2348/69 (O127:H6), which is a wild-type strain isolated from an infant with diarrhea (26), was generously provided by B. B. Finlay (University of British Columbia, Vancouver, British Columbia, Canada). In each experiment, bacteria which were stored as frozen stock cultures at 270°C were grown overnight at 37°C on tryptic soy agar plates (Difco, Detroit, Mich.). An isolated bacterial colony was then inoculated into tryptic soy broth (TSB) and incubated for 16 h, without shaking, at 37°C under normal atmospheric condi- tions for use in experiments the following day. Preparation of HEp-2 cell monolayers. HEp-2 cells (CCL-23) were obtained from the American Type Culture Collection (Rockville, Md.). The cells were grown at 37°C in a humidified atmosphere of 5% CO2–95% air in minimal essential medium supplemented with 10% fetal bovine serum (FBS). Subconflu- ent monolayers were prepared for the binding assays by disrupting HEp-2 mono- layers with a solution of 0.25% (vol/vol) tissue culture-grade trypsin in FC buffer (0.14 M NaCl, 5.0 M mM KCl, 20.0 mM Tris-HCl, 5.0 mM Tris base, 0.5 mM EDTA [pH 7.2]). After the trypsinized HEp-2 cells were suspended in fresh tissue culture medium, approximately 7.5 3 103 cells in 150 ml of culture medium were added to individual wells, with each well containing a 6-mm-diameter removable polystyrene disk (Biomedical Workshop, University of Alberta, Ed- monton, Alberta, Canada) covering its bottom, of 96-well tissue culture plates. The plates were then incubated in a CO2 incubator until the next day. EPEC cell binding assay. For all experiments, EPEC E2348/69 was cultured in Dulbecco modified Eagle medium (DMEM) (catalog number 23800; Gibco, Burlington, Ontario, Canada) supplemented with 44 mM NaHCO3, 40 mM phenol red, and 25 mM glucose, which was pre-equilibrated overnight in a CO2 incubator (42). FBS was not included in this culture medium. Prior to each experiment, 40 ml of the TSB-grown bacteria were inoculated into 4 ml of DMEM in borosilicate glass culture tubes (15 by 75 mm), which were then incubated for 1 h in a CO2 incubator to induce the expression of EPEC virulence factors (32, 42, 44). To determine the optimum concentration of glycoconjugates for the binding inhibition experiments, 32 ml of DMEM bacterial culture (ca. 3 3 106 to 5 3 106 CFU) and 52 ml of DMEM (pre-equilibrated) were added to empty wells of a 96-well microtiter plate. Next, 16 ml of BSA-glycoconjugate solution (undiluted [5 mg/ml] or diluted 1:2 or 1:4 in PBS) was added to the wells to obtain final inhibitor concentrations of 0.8, 0.4, or 0.2 mg/ml, respectively. For all subsequent experiments, the volumes were adjusted such that 32 ml of DMEM bacterial culture, 56 ml of DMEM, and 12 ml of BSA-glycoconjugate (final concentration, 0.6 mg/ml) were added to wells of a 96-well plate. The plate was then incubated at 37°C in a CO2 incubator for 30 min, after which time the entire contents from each of the wells were transferred to wells containing subconfluent HEp-2 cell monolayers from which the culture medium was first removed. After this microtiter plate was incubated for an additional 30 min at 37°C in the CO2 incubator, the cells were washed three times with PBS, fixed with methanol for 10 min, and stained with Giemsa stain for 20 min. The polystyrene disks were removed from the microtiter plate wells, and EPEC adherence was monitored microscopically by using a 3100 objective lens. A total of 150 to 200 randomly chosen HEp-2 cells were examined, and those having attached microcolonies consisting of five or more bacteria were considered positive for LA EPEC (44). Effect of glycoconjugate preincubation period on bacterial attachment. EPEC E2348/69 was grown in DMEM for 1 h in a CO2 incubator as described above. Next, 32 ml of bacterial culture and 56 ml of DMEM were added to empty wells of a 96-well tissue culture plate. At this time, 12 ml of BSA-glycoconjugate (final concentration, 0.6 mg/ml per well) was added to sample wells for which the effect of preincubation with the BSA-glycoconjugate(s) was to be determined. After the plate was incubated in a CO2 incubator for 30 min, 12 ml of BSA-glycocon- jugate was added to the remaining, nonpreincubated sample wells. The contents of all wells were then immediately transferred to wells of a 96-well microtiter plate containing subconfluent HEp-2 cell monolayers grown on polystyrene disks, and the plate was returned to a CO2, 37°C incubator for 30 min. The cells were then washed three times with PBS, fixed with methanol, and stained with Giemsa stain. The percentage of HEp-2 cells with LA EPEC was determined as described above. Determination of BfpA and intimin expression. After EPEC E2348/69 was incubated in DMEM for 1 h to induce the expression of virulence factors, 96 ml of bacterial culture, 168 ml of DMEM, and 36 ml of BSA-glycoconjugate (final concentration, 0.6 mg/ml per well) were added to empty wells of a 48-well tissue culture plate. The plate was incubated in a CO2 incubator for 1 h before the contents of each well were transferred to microcentrifuge tubes. The bacteria were washed once with PBS by centrifugation, and the resulting pellet was lysed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sam- ple buffer containing 50 mM dithiothreitol (24). Bacterial proteins were sepa- rated by SDS-PAGE (12.5% polyacrylamide) and electrophoretically transferred to an Immobilon-P membrane (Millipore, Bedford, Mass.). Since different antibodies were used to detect specific proteins, the membrane was cut into three sections by using the prestained molecular size standards as a guide. Nonspecific binding sites of the membranes were blocked with 5% (wt/vol) skim milk in PBS containing 0.05% Tween 20 (PBST), and the sections were incubated with either anti-intimin (1:2,000 dilution), anti-MBP (1:10,000 dilu- tion), or anti-BfpA (1:4,000 dilution) antibodies for 2 h at room temperature. The membranes were then washed three times with PBST and incubated with goat anti-rabbit peroxidase-conjugated antibodies (1:18,000 dilution) for 1.5 h at room temperature. After being washed again with PBST, followed by three washes with PBS, the membranes were incubated with the enhanced chemilu- minescence (ECL) color development reagents according to the manufacturer’s instructions (Amersham, Oakville, Ontario, Canada). Protein bands were visu- alized by exposing the membranes to Kodak X-Omat Blue XB-1 film. Bands corresponding to intimin, MBP, and BfpA in each sample were analyzed by using an LKB Ultroscan XL laser densitometer supplied with an LKB 2220 integrator. The amounts of intimin and BfpA in each sample were normalized to the amount of the internal standard, MBP, in each gel lane. Statistical analysis. The significance of any differences in EPEC LA or in the levels of BfpA and intimin was determined by using the nonparametric Mann- Whitney U test. RESULTS Inhibition of EPEC LA by the BSA-glycoconjugates. Prelim- inary experiments were performed to determine the concen- tration of glycoconjugate(s) to be used in experiments. The results of these dose-dependent binding inhibition experiments demonstrated that LacNAc-BSA was the most effective inhib- itor of EPEC E2348/69 attachment to HEp-2 cells (Fig. 1). This inhibition was concentration dependent over the range examined, i.e., 0.8, 0.4, or 0.2 mg of LacNAc-BSA per ml, resulting in approximately 87, 65, and 40% reductions, respec- tively, in EPEC LA to HEp-2 cells. Based on the inhibitory effects observed with LacNAc-BSA in Fig. 1, we used the BSA-glycoconjugates at a final concen- tration of 0.6 mg/ml in all subsequent experiments. To confirm the appropriateness of this concentration, three additional ex- periments were performed with EPEC E2348/69 (Fig. 2). Sim- ilar to the results presented in Fig. 1, LacNAc-BSA inhibited EPEC attachment to the greatest extent. LeX-BSA was the TABLE 1. Oligosaccharide sequences of BSA-glycoconjugates Oligosaccharide Structure LacNAc........................Galb(134)GlcNAc LeX ..............................Galb(134)[Fuca (133)]GlcNAc LeY ..............................Fuca(132)Galb(134)[Fuca(133)]GlcNAc VOL. 67, 1999 EFFECT OF LACTOSYL STRUCTURES ON EPEC VIRULENCE FACTORS 3303 second most effective inhibitor of EPEC binding in these ex- periments, with LeY-BSA being the least effective. Compara- ble results were observed in later experiments performed in a similar manner (see Fig. 5). A statistical analysis of the com- bined data from these experiments indicated that, of the gly- coconjugates, only LacNAc-BSA significantly inhibited EPEC binding to HEp-2 cells compared to bacteria which were incu- bated with BSA (P 5 0.007). LacNAc-BSA inhibited EPEC binding significantly more than LeX-BSA and LeY-BSA (P , 0.037), whereas there was no significant difference between the inhibitory effects of LeX-BSA and LeY-BSA (P 5 0.201). Additional control experiments confirmed that the inhibitory effect of these glycoconjugates was not due to a toxic effect of these compounds on the bacteria (data not shown). Effect of preincubation on EPEC attachment. The proce- dure used in the glycoconjugate inhibition binding experiments involved preincubating EPEC E2348/69 with various glycocon- jugates for 30 min prior to adding the mixtures to the HEp-2 cell monolayers. This preincubation step was performed on the assumption that the multivalent BSA-glycoconjugate inhibitors required a finite amount of time to occupy bacterial adhesin receptor binding sites prior to exposing the EPEC-glycoconju- gate mixtures to the HEp-2 cells. This assumption would pre- dict that the BSA-glycoconjugate inhibitors might be less ef- fective if this preincubation step was omitted. To test this prediction, experiments were performed in which the bacteria were either preincubated for 30 min with LacNAc-BSA before being added to the HEp-2 cells or were added simultaneously with LacNAc-BSA to the monolayers. As shown in Fig. 3, eliminating the preincubation step resulted in an approxi- mately twofold decrease in the inhibitory activity of LacNAc- BSA, although this difference was not statistically significant (P 5 0.121). Without preincubation, EPEC LA to HEp-2 cells FIG. 1. Optimization of BSA-glycoconjugate(s) concentration for experiments. DMEM-grown EPEC E2348/69 were preincubated with various concentrations of different BSA-glycoconjugate(s) and then added to tissue culture wells containing subconfluent monolayers of HEp-2 cells. After bacterial binding was allowed to occur, the cells were washed, fixed with methanol, and stained with Giemsa stain. HEp-2 cells with adherent microcolonies consisting of five or more bacteria were considered positive for LA EPEC. Two independent trials were performed, trial 1 (F) and trial 2 (E), with each point representing a single determination for each sample. Bars indicate the average(s) of the data from both trials. FIG. 2. Inhibition of EPEC E2348/69 binding to HEp-2 cells with BSA- glycoconjugates at a final concentration of 0.6 mg/ml per well. Experiments were performed essentially as described in the legend to Fig. 1 except that bacteria were preincubated with BSA-glycoconjugates present at final concentrations of 0.6 mg/ml per well. Data points for trial 1 (F), trial 2 (‚), and trial 3 (E) are indicated. Error bars for each of these points represent the range in values obtained for duplicate samples. The overlaying bar graph indicates the mean(s) of the data obtained in all three trials. FIG. 3. Effect of preincubation period on EPEC E2348/69 attachment to HEp-2 cells. The glycoconjugates (final concentration, 0.6 mg/ml per well) were