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Basis for N-acetyllactosamine-mediated inhibition of enteropathogenic Escherichia coli localized adherence

¹ Department of Microbiology and Infectious Diseases, Faculty of Medicine, University of Calgary, 3330 Hospital Drive, NW, Calgary, AB, T2N 4N1, Canada

² Department of Chemistry, University of Alberta, Edmonton, AB, T6G 2G2, Canada

³ Alberta Research Council, 250 Karl Clark Road, Edmonton, AB, T6N 1E4, Canada

⁴ Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, AB, T6G 2H7, Canada

Correspondence
Glen D. Armstrong
glen.armstrong{at}ucalgary.ca

Received 23 September 2005
Accepted 19 February 2006

Abbreviations: BFP, bundle-forming pili; EPEC, enteropathogenic Escherichia coli; LA, localized adherence; LacNAc, N-acetyllactosamine; MBP, maltose binding protein; Pk, Gal(1,4)ßGal(1,4)ßGlc.

	INTRODUCTION

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
Enteropathogenic Escherichia coli (EPEC) serotypes cause neonatal diarrhoea in developing nations (Hart et al., 1993) and occasional diarrhoea in adults who travel to endemic regions of the world (Bieber et al., 1998; Hart et al., 1993). EPEC infect and colonize their hosts through a highly interactive process thought to involve three steps: initial binding, signal transduction, and intimate adherence (Campellone & Leong, 2003; Delahay et al., 2001; Donnenberg & Kaper, 1992; Donnenberg & Whittam, 2001; Kenny, 2002). In the first stage of this process, EPEC form micro-colonies on the host-cell surface through a process known as localized adherence (LA) (Donnenberg et al., 1992; Giron et al., 1993). EPEC micro-colony formation during LA is dependent on rope-like surface appendages called bundle-forming pili (BFP) (Donnenberg et al., 1992; Giron et al., 1993). While the role of BFP in the actual adherence of EPEC to the host cell is unknown, human volunteer studies have shown that BFP are required for full EPEC virulence in adults (Bieber et al., 1998). BFP have also been shown to be required for efficient adherence of EPEC to intestinal brush border cells during the early stages of infection (Cleary et al., 2004). Moreover, Tobe and Sasakawa (2001) have demonstrated a clear requirement for BFP both in the initial attachment of EPEC to human intestinal tissue culture Caco-2 cell surfaces and in LA to this cell line. This group have also demonstrated that isolated BFP attach to Caco-2 cells, and it appears that contact with the Caco-2 cell surface induces EPEC to shed their BFP, concomitant with micro-colony dispersal and intimate adherence.

EPEC serotypes have been shown to bind to complex carbohydrate receptor sequences displayed on host-cell glycolipids and glycoproteins (Cravioto et al., 1991; Idota & Kawakami, 1995; Jagannatha et al., 1991; Vanmaele et al., 1999). Previous research in our laboratory has demonstrated that EPEC lose their BFP by an undetermined mechanism when the organisms are exposed to synthetic N-acetyllactosamine (LacNAc) glycoconjugates, the same glycoconjugates which we have found to inhibit EPEC LA to HEp-2 cells (Vanmaele et al., 1999). These findings reflect the Caco-2 cell-membrane-mediated BFP-shedding events described by Tobe and Sasakawa (2001). Clearly, the expression, regulation, and precise function of BFP in EPEC colonization and pathogenesis in the human intestine remain enigmatic.

Regardless of the continuing debate surrounding the regulation and function of BFP in EPEC colonization in vivo, the studies reported herein were designed to further investigate the interaction of EPEC with complex carbohydrate sequences, as described in our previous reports on the subject.

	METHODS

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
Reagents and bacterial strains. The wild-type EPEC strain E2348/69 (serotype O127 : H6), initially isolated from an infant with diarrhoea, was provided by Dr M. Finlayson (Provincial Laboratory of Alberta, Edmonton, AB). JPN15, a spontaneous EAF plasmid-cured derivative of E. coli E2348/69, was provided by Dr B. Brett Finlay (University of British Columbia, Vancouver, BC). The non-polar bfpF insertion mutant UMD916 and complemented (UMD916 containing pMSD217) EPEC strains (Anantha et al., 1998) were provided by Dr M. Donnenberg (University of Maryland School of Medicine, Baltimore, MD). These bacteria were routinely cultivated on conventional tryptic soy agar (TSA) plates at 37 °C to obtain individual colonies. Rabbit BFP-specific antiserum was provided by Dr J. A. Giron (University of Arizona, Tucson, AZ). The rabbit maltose binding protein (MBP) antiserum was purchased from New England Biolabs. Goat anti-rabbit IgG conjugated to 6 nm gold particles was purchased from Cedarlane.

All the glycoconjugates listed in Table 1 consisted of chemically synthesized 8-methoxycarbonyloctyl (MCO) glycosides (Lemieux et al., 1977) covalently coupled to BSA. The conjugation ratios (N, mole MCO glycoside sequence per mole BSA) were determined by mass spectroscopy analysis. The glycoconjugates were dissolved in distilled water to a final concentration of 5 mg ml^–1.

Preparation of tissue culture cell monolayers and LacNAc–BSA LA binding inhibition assays. The HEp-2 (CCL-23), Caco-2 (HTB-37) and T84 (CCL-248) cell lines were obtained from and propagated according to the American Type Culture Collection recommendations. Cell monolayers were prepared in 96-well plates containing polystyrene discs, as described in our previous article (Vanmaele et al., 1999).

Prior to each experiment, BFP expression was induced by inoculating 10 µl of a tryptic soy broth (TSB) bacterial culture, grown statically overnight at 37 °C from a single colony picked from a TSA plate, into 1 ml Dulbecco modified Eagle medium (DMEM, Gibco) which had been pre-equilibrated overnight in a CO₂ incubator and supplemented with 44 mM NaHCO₃, 40 µM phenol red and 25 mM glucose (Vanmaele et al., 1995, 1999). After 30 min at 37 °C in the CO₂ incubator, these cultures were added to 96-well plates containing the BSA glycoconjugates and incubated for an additional 30 min at 37 °C in a CO₂ incubator. The contents of each of these wells were then transferred to the corresponding wells of a 96-well microtitre plate containing the subconfluent HEp-2, Caco-2 or T84 cell monolayers on polystyrene discs. After 30 min at 37 °C in the CO₂ incubator, the tissue culture cells were washed three times with physiological PBS, pH 7.2. HEp-2 cells were fixed with methanol for 10 min and then stained with Giemsa stain for 20 min. The polystyrene discs were removed from the microtitre plate wells, and EPEC LA was quantified microscopically as described previously (Vanmaele et al., 1999).

Since individual cells could not be distinguished, it was not possible to enumerate EPEC LA to Caco-2 and T84 cell monolayers by the same microscopic method used to determine EPEC LA to Hep-2 cells. Therefore, EPEC attachment to these cell lines was determined by solubilizing the washed monolayers at 37 °C for 30 min in 100 µl PBS containing 0.01 % (v/v) Triton X-100. The solubilized cells were then serially diluted in sterile PBS and spread on TSA plates. After incubation overnight at 37 °C, c.f.u. were enumerated to determine the number of adherent EPEC per individual monolayer. In addition, one disc from each Caco-2 and T84 LA inhibition assay was fixed, stained and observed microscopically as described above to confirm the LA phenotype of the adherent organisms (Vanmaele et al., 1999).

LacNAc–BSA-mediated reduction of BfpA, as assessed by immunoblotting. Overnight TSB cultures of all the strains used in this study were incubated in DMEM to induce BFP expression (Vanmaele et al., 1999). These cultures were then mixed with BSA, LacNAc–BSA, Pk–Au or LacNAc–Au at a concentration of 0.4 mg ml^–1 in DMEM and incubated at 37 °C for an additional 30 min in the CO₂ incubator. After centrifugation at 14 000 g for 10 min, the supernatant solutions were removed and the resulting bacterial cell pellets were dissolved in SDS sample buffer containing 50 mM DTT and subjected to conventional one-dimensional SDS-PAGE using 12.5 % (v/v) separating gels. The separated proteins were then electrophoretically transferred to Immobilon-P (Millipore) membranes, and the immunoreactive BfpA and MBP (an invariant E. coli housekeeping protein used to control for sample-loading inconsistencies) bands were subsequently detected as per the procedure described in our previous article (Vanmaele et al., 1999). The resulting immunoblots were subjected to densitometry analysis using the Kodak Image Station 2000MM and associated Kodak ID image analysis software (version 3.6.5). Sample-loading inconsistencies were then corrected for by normalizing the densitometry intensities of each of the BfpA bands in each gel lane to the intensity of the accompanying invariant E. coli MBP band.

Immunogold and gold nanoparticle labelling procedures. BFP were isolated for these studies as described elsewhere (Giron et al., 1991). The bacteria for these studies were grown at 37 °C overnight on TSA plates supplemented with 5 % (v/v) defimbrinated sheep blood to induce BFP expression (Giron et al., 1991). Single bacterial colonies were then picked for analysis. These were gently suspended in 200 µl monoethanolamine (Sigma-Aldrich) buffer (pH 10.0). Twenty microlitres of each of the suspensions were subsequently applied onto Formvar and carbon-coated copper grids (Cedarlane). These were incubated for 10 min at 4 °C, after which time excess fluid was gently wicked from the grids. All remaining steps in the procedure were performed at 4 °C, a condition chosen to prevent BFP retraction. Immunogold labelling was performed by floating the grids, sample side down, for 20 min on a 50 µl drop of PBS containing 5 % (w/v) BSA. The grids were then exposed for 1 h to rabbit anti-BFP serum diluted 100-fold in PBS. After a thorough washing with PBS, the grids were incubated for an additional 30 min in a solution containing goat anti-rabbit IgG conjugated to 6 nm gold particles, diluted 30-fold in PBS. The grids were again washed thoroughly with PBS, stained with 1.0 % (w/v) phosphotungstic acid (PTA) for 5 min and gently wicked dry. The glycoside–gold nanoparticle labelling procedure was performed by floating the grids, sample side down, for 45 min on a 50 µl drop of either LacNAc–Au or Pk–Au suspended at a concentration of 10 mg ml^–1 in PBS. The grids were then thoroughly washed with PBS and stained using PTA, as described above. Finally, all the grids were examined using a Hitachi S-7000 transmission electron microscope.

	RESULTS AND DISCUSSION

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
Screening of potential EPEC LA inhibitors

In the present study, we extended our screening of potential EPEC glycan receptors reported in our previous article (Vanmaele et al., 1999) to include the synthetic glycoconjugates listed in Table 1. These all consisted of LacNAc, or modified LacNAc, sequences containing various fucosyl, sialyl or sulfo substitutions and additions. The outcome of this screening assay confirmed our previous finding (Vanmaele et al., 1999) that LacNAc–BSA is the most significant (P<0.001, Student's t test) glycan-based inhibitor of LA (data not shown). At a concentration of 0.8 mg ml^–1, LacNAc–BSA caused a 98.5 % (±1.72 %) reduction in the number of HEp-2 cells with LA EPEC compared to the BSA control. LacNAc–BSA was followed in relative inhibitory potency by linear B (IV) (54.3±3.25 % reduction of LA EPEC) and sialyl-Lewis A (49.5±11.0 % reduction of LA EPEC) glycoconjugates, both of which significantly inhibited EPEC LA (P<0.005) at a concentration of 0.8 mg ml^–1. The remaining glycoconjugates listed in Table 1 inhibited LA weakly or not at all. We therefore focused the remainder of the present study on further investigating the interaction of EPEC with LacNAc.

EPEC binding inhibition experiments were also performed using LacNAc glycosides coupled to 5 nm gold nanoparticles. LacNAc–Au, at a concentration of 0.8 mg ml^–1, inhibited LA to HEp-2 cells by 73.2±7.89 % (Fig. 1). In contrast, gold nanoparticles containing the Pk glycan sequence had no inhibitory effect on LA relative to BSA alone (Fig. 1). Therefore, LacNAc–BSA and LacNAc–Au both specifically inhibit EPEC LA to HEp-2 cells. These observations confirm LacNAc as the active inhibitory component in the two different glycoconjugate preparations.

View larger version (14K): [in this window] [in a new window]   Fig. 1. LacNAc–Au nanoparticle-mediated inhibition of EPEC E2348/69 LA to HEp-2 cells. EPEC were pre-incubated at 37 °C in DMEM to induce BFP expression and subsequently with 0.8 mg ml–1 LacNAc–BSA, BSA, LacNAc–Au or Pk–Au. LA was determined as described previously (Vanmaele et al., 1999). The data represent the means and standard errors of three trials per inhibitor. The difference between the inhibitory capacity of LacNAc–BSA versus BSA was significant, as was that between the inhibitory capacity of LacNAc–Au versus Pk–Au or BSA (P<0.001, Student's t test).

View larger version (20K): [in this window] [in a new window]   Fig. 2. LacNAc–BSA-mediated inhibition of E2348/69 LA to HEp-2, Caco-2 and T84 cells. The data represent the means and standard errors of three trials per inhibitor. The difference between the inhibitory capacity of LacNAc–BSA versus BSA was significant in all three cell lines tested (P<0.001, Student's t test). Black bars, HEp-2 cells; white bars, Caco-2 cells; grey bars, T84 cells.

View larger version (21K): [in this window] [in a new window]   Fig. 3. Inhibition of LA to HEp-2 cells by 0.8 mg ml–1 LacNAc–BSA or LacNac–Au in wild-type E2348/69, the bfpF insertional mutant strain (UMD916), or UMD916 complemented with a functional bfpF gene expressed from pMSD217. Experiments were performed in triplicate, and the error bars indicate the standard deviation from the mean. Also presented are the results of immunoblotdetection of BfpA and MBP in these strains after exposure to either 0.4 mg ml–1 BSA or LacNac–BSA (a) or Pk–Au or LacNAc–Au (b) for 30 min at 37 °C. The individual immunoblots from each experiment were subjected to densitometry analysis, thereby allowing the intensity of the BfpA band in each lane to be normalized to that of the accompanying MBP band, an invariant housekeeping protein in E. coli. The resulting averaged data are presented quantitatively in panels (a) and (b) and the error bars indicate the range of the results from the two independent experiments. The immunoblotting experiment was performed twice with the BSA conjugates and in triplicate with the gold conjugates. Also presented in panel (c) is a representative immunoblot experiment from one of the experiments employing the gold nanoparticle glycoconjugates. Lanes 1 indicate EPEC incubated with 0.4 mg LacNAc–Au ml–1; lanes 2 indicate EPEC incubated with 0.4 mg Pk–Au ml–1.

The opposing mechanisms whereby Caco-2 plasma membranes and LacNAc–BSA promote the loss of BFP from wild-type EPEC might be explained by the physical state of the two different sources of receptors. In this regard, the bacteria may not be able to retract their BFP if these are bound to insoluble Caco-2 membranes. Rather, the membrane-bound BFP filaments might be physically separated from the bacterial surface when the BfpF-mediated retraction system is activated. These BFP would therefore not be available to the degradation mechanisms present within the bacterial outer membrane or periplasmic compartments, and would remain antigenically intact in the Caco-2 membrane fraction. By contrast, the soluble LacNAc–BSA preparation might not present such a physical impediment to BFP retraction and subsequent BfpA degradation, perhaps by the EPEC Cpx-DegP envelope stress-response system (Raivio, 2005), within the bacterial cell.

Leverton and Kaper (2005) have recently reported that transcription of the bfpA gene increases in EPEC after the organisms have been in contact with HEp-2 cells for 3 h. Moreover, BfpA is also detected in Western immunoblots of EPEC-infected HEp-2 cells at both 3 and 5 h after bacterial attachment. In our experiments, the presence of BfpA was determined 30 min after the bacteria were exposed to LacNAc–BSA. It is possible, therefore, that the initial binding of BFP to host-cell glycan receptors may induce their rapid retraction and degradation, without affecting transcription of the bfpA gene. BFP retraction might serve to bring the bacteria into close apposition with the host-cell membrane, thereby expediting the microvillus effacement and intimate adherence phase of the EPEC colonization process. Following these early binding events, BFP expression may be upregulated, as reported by Leverton and Kaper (2005), as the EPEC prepare to perpetuate the colonization process in the infected host.

Identification of the EPEC LacNAc-specific adhesin

To identify the LacNAc-specific adhesin on the surface of EPEC, E2348/69 and UMD916 cells were exposed to LacNAc or Pk–Au nanoparticles at 4 °C (Fig. 4). In these experiments, LacNAc–Au nanoparticles were only observed in association with BFP (Fig. 4a). No gold nanoparticles were observed in association with the bacterial cell surface (Fig. 4c). Additionally, LacNAc–Au nanoparticles were observed in association with BFP purified from the UMD916 EPEC strain (Fig. 4b). Whereas the Pk–Au nanoparticle preparation neutralized Shiga toxin 1 in the Verocytotoxicity assay (data not shown), thereby confirming the presence and functional integrity of the Pk glycan sequences in the preparation (Marcato et al., 2001), no Pk–Au nanoparticles were observed on either the BFP or the cell surfaces of E2348/69 (Fig. 4d) and UMD916 (Fig. 4f). The identity of BFP in these micrographs was confirmed by immunogold labelling using BFP-specific rabbit antiserum (Fig. 4e). Further, BFP, LacNAc–Au and Pk–Au nanoparticles were not detected on BFP-negative JPN15 cells (data not shown).

View larger version (121K): [in this window] [in a new window]   Fig. 4. Transmission electron microscopic examination of LacNAc–Au binding to BFP. Panels: (a) BFP-expressing wild-type EPEC strain E2348/69; (b) BFP purified from EPEC strain UMD916; (c) EPEC strain UMD916; (d) BFP-expressing E2348/69 incubated with Pk–Au; (f) strain UMD916 incubated with Pk–Au. BFP were also identified by immunogold labelling: panel (e). All samples were stained with 1.0 % (w/v) phosphotungstic acid, pH 7.2, and were visualized using a Hitachi S-7000 transmission electron microscope. Bars, 100 nm.

In sum, the data presented herein are consistent with a model in which EPEC employ their BFP to initially attach themselves to LacNAc-like glycan receptors on host-cell surfaces. Although the intimate stages of the EPEC colonization process can occur in the absence of BFP in vitro (Hicks et al., 1998), a BfpF-mediated retraction system which can pull the BFP-tethered bacteria closer to the host-cell plasma membrane may be needed to expedite the intimate adherence phase of the EPEC colonization process in vivo. If so, innovative intervention strategies devised to either competitively inhibit BFP-mediated binding to host-cell glycan receptors or antagonize the BfpF-mediated retraction mechanism could be of therapeutic benefit in patients suffering from EPEC-mediated diarrhoea.

	ACKNOWLEDGEMENTS

 
Financial and material support was from the Alberta Research Council (O. P. S.), Canadian Bacterial Diseases Network (G. D. A.), the Alberta Ingenuity Centre for Carbohydrate Science (G. D. A.) and the Canadian Institutes for Health Research (CIHR) (G. D. A.). R. M. H. was the recipient of a Studentship from the National Sciences and Engineering Research Council of Canada (NSERC). P. M. was the recipient of Doctoral Studentships from the CIHR and Alberta Heritage Foundation for Medical Research.

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