Department of Pediatrics, Harvard Medical School, and Gastrointestinal Cell Biology Laboratory, Children's Hospital, Boston, Massachusetts 02115
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ABSTRACT |
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The initial step in many mucosal
infections is pathogen attachment to glycoconjugates on the apical
surfaces of intestinal epithelial cells. We examined the ability of
virus-sized (120-nm) and bacterium-sized (1-µm) particles to adhere
to specific glycolipids and protein-linked oligosaccharides on the
apical surfaces of rabbit Peyer's patch villus enterocytes,
follicle-associated enterocytes, and M cells. Particles coated with the
B subunit of cholera toxin, which binds the ubiquitous glycolipid GM1,
were unable to adhere to enterocytes or M cells. This confirms that
both the filamentous brush border glycocalyx on enterocytes and the
thin glycoprotein coat on M cells can function as size-selective
barriers. Oligosaccharides containing terminal (1,4)-linked
galactose were accessible to soluble lectin Ricinus communis
type I on all epithelial cells but were not accessible to lectin
immobilized on beads. Oligosaccharides containing
(2,3)-linked
sialic acid were recognized on all epithelial cells by soluble
Maackia amurensis lectin II (Mal II). Mal II coated 120-nm (but
not 1-µm) particles adhered to follicle-associated enterocytes and M
cells but not to villus enterocytes. The differences in receptor
availability observed may explain in part the selective attachment of
viruses and bacteria to specific cell types in the intestinal mucosa.
Peyer's patch; glycocalyx; adhesion; pathogen; M cells
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INTRODUCTION |
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ENTERIC PATHOGENS SELECTIVELY infect specific epithelial cell types in the gastrointestinal tract (1, 6, 25). For example, rotavirus replicates within villus enterocytes (39), whereas reovirus selectively binds to, and is endocytosed by, M cells (51). Adherence, the initial step in the infection process, is postulated to be mediated by lectinlike microbial adhesins that recognize defined glycoproteins and/or glycolipid epitopes on the apical surfaces of intestinal epithelial cells (6, 22, 25, 27). Lectinlike adhesins have been identified on a wide array of enteroviruses and pathogenic bacteria (1, 6, 19, 27). For example, rotavirus binds the glycosphingolipid asialo-GM1 and O-linked sialylglycoproteins in overlay assays (49, 50), and reovirus attachment to mouse fibroblasts in vitro depends on the presence of host cell sialoglycoconjugates (14). However, because most of these glycolipids and galactosyl- or sialic acid-containing oligosaccharides are widely distributed on intestinal epithelial cells, receptor distribution alone cannot explain the cell type-specific tropisms of viral and bacterial pathogens that are observed in vivo. Thus additional factors must exist that influence pathogen attachment to specific cells or regions of the gastrointestinal mucosa (26, 27).
The intestinal epithelium is comprised primarily of absorptive villus enterocytes (28). Enterocyte apical surfaces in vivo are highly differentiated structures consisting of rigid, closely packed microvilli whose membranes contain stalked glycoprotein enzymes (33, 45). In addition, the tips of enterocyte microvilli are coated with a 400- to 500-nm-thick meshwork called the filamentous brush border glycocalyx (FBBG) (23), which is composed of highly glycosylated transmembrane mucins (29, 30). The epithelium overlying organized mucosal lymphoid nodules, the so-called follicle-associated epithelium (FAE), is comprised of enterocyte-like cells interspersed with M cells. The M cell is a morphologically distinct epithelial cell type whose primary function is the transport of macromolecules, particles, and microorganisms from the lumen to underlying lymphoid tissue (11, 35). M cells generally lack an organized brush border and a well-defined FBBG, although their apical membranes have a thin (20-30 nm) glycoprotein coat (10).
We have recently shown that the FBBG on the apical surfaces of rabbit villus and FAE enterocytes is a size-selective barrier that can prevent particles from gaining access to membrane glycolipids (10). This was demonstrated using the B subunit of cholera toxin (CTB), which specifically binds to ganglioside GM1, a glycolipid whose carbohydrate head extends just 2.5 nm from the plasma membrane (47). When applied in soluble form to rabbit mucosa, CTB (diameter 6.8 nm) had free access to GM1 on all epithelial cell types and bound to villus enterocytes, FAE, and M cells (10). When CTB was coupled to colloidal gold to form a particulate probe 28 nm in diameter, it was no longer able to penetrate the FBBG and was therefore unable to bind to enterocytes, but the particles did adhere to the apical surfaces of M cells. The thin glycoprotein coat on M cells can prevent larger particles from gaining access to ganglioside GM1, however, because CTB-coated, 1-µm particles were unable to bind. Our previous studies did not test whether the M cell glycoprotein coat can exclude particles in the size range of most viruses from contact with membrane glycolipids in vivo. This is important because it has been shown that HIV-1 (120 nm in diameter) attaches to intestinal cells in culture via galactosylceramide (9, 12). HIV can adhere to M cells in rabbit intestine (2) and may possibly exploit M cell transport activity to enter the rectal mucosa in humans (34).
Protein-linked oligosaccharides would be expected to provide more accessible binding sites for microorganisms because they extend up to 500 nm from enterocyte plasma membranes and 20-30 nm from M cell membranes (10). They would thus be the first structures encountered by intestinal pathogens (25). Integral membrane mucins such as those of the enterocyte FBBG consist of up to 80% heterogeneous O-linked carbohydrates that form dense, negatively charged gels thought to function in cytoprotection (48). Oligosaccharide side chains on both mucin and nonmucin membrane glycoproteins are notoriously heterogeneous, and the exact composition, structure, and topology of only a small minority have been defined (25). There is evidence that glycosylation patterns not only of M cells but of the entire FAE differ from those of villus epithelium (5, 13, 17, 18, 46), but the exact topology or accessibility of specific oligosaccharide structures on epithelial cells of small intestinal villi and FAE has not been explored. Such information could be important for understanding microbial tropism in the intestine and for designing strategies for targeting of particulate vaccines and vaccine vectors to the FAE and M cells.
For this study we selected three ligands that recognize glycolipid or
protein-linked oligosaccharide epitopes that are known to be exploited
by enteric pathogens. We examined the ability of the ligands
immobilized on virus-sized (120-nm) and bacterium-sized (1-µm)
particles to adhere to apical surfaces of rabbit Peyer's patch villus
enterocytes, FAE, and M cells. Particles were coated with CTB to probe
accessibility of the glycolipid GM1, the lectin Ricinus
communis agglutinin type I (RCA-I) specific for terminal (1,
4)-linked galactose, and Maackia amurensis lectin II (Mal II),
which recognizes a hierarchy of oligosaccharides containing
(2,3)-linked sialic acid. Data presented indicate that the
relatively thin glycoprotein coat on the apical surfaces of M cells is
sufficient to prevent virus-sized particles from gaining access to
glycolipids like GM1. In addition, we found that protein-linked
oligosaccharides vary in their accessibility to particulate ligands and
that differences in accessibility exist between villus and FAE. We
conclude that successful attachment of pathogens to glycolipids and
glycoproteins on apical surfaces of intestinal epithelial cells is
dependent on both receptor availability and receptor accessibility.
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MATERIALS AND METHODS |
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Chemicals and reagents. All lectins, lectin derivatives, and fluorophore-conjugated streptavidin reagents were purchased from Vector Laboratories (Burlingame, CA). Biotinylated CTB was purchased from List Laboratories (Campbell, CA). BSA, biotinylated BSA, and biocytin were from Sigma (St. Louis, MO). Neutravidin-coated, fluorescent, carboxylate-modified polystyrene particles (FluoSpheres) were purchased from Molecular Probes (Eugene, OR). Liquid paraformaldehyde (16% solution) was obtained from Electron Microscopy Sciences (Fort Washington, PA).
Preparation of ligand-coated particles. The fluorescent red or green virus-sized particles used in this study had an estimated diameter of 0.093 µm ± 7.5%. The manufacturer coated them with Neutravidin, resulting in ~10 nmol biotin binding sites per milligram of polystyrene. To derivitize particles with ligands, 1 × 1012 particles were suspended in 500 µl of PBS (50 mM sodium phosphate, pH 7.4, 50 mM NaCl) containing 0.02% Tween-20, 0.5% BSA, 2 mM NaN3, and 50 µg/ml of biotinylated ligand or biocytin. The particle mixtures were gently rocked overnight at 4°C and then transferred to a 0.5-ml Dispodialyzer dialysis tube (Spectrum, Houston, TX) with a molecular weight cut-off of 300,000. Excess unbound ligand was removed by dialysis against 4 l of PBS containing 2 mM NaN3 changed daily for 4 days. In control experiments, this dialysis protocol was sufficient to remove >95% of soluble ligand. After dialysis, particles were transferred to siliconized microcentrifuge tubes and stored at 4°C in the dark. The bacterium-sized (1.0 µm ± 2.5%) particles were coated with biotinylated ligand as described previously (10). Briefly, particles and biotinylated ligand (400 µg/ml) were combined in 1 ml of PBS and incubated with gentle rocking overnight at 4°C. The particles were then collected by centrifugation (3,500 g) and washed with PBS several times to remove excess, unbound ligand. The particles were resuspended in 400 µl PBS containing 2 mM NaN3 and then stored in the dark at 4°C. Before use in adhesion assays, particles were vortexed for 2 min at room temperature and then bath sonicated (Laboratory Supply, Hicksville, NY) in an ice slurry for 10 min. Ligand-coated, fluorescent green particles and biocytin-quenched (or BSA-coated), fluorescent red particles were diluted at a 1:1 ratio as determined by fluorescence microscopy into sterile PBS containing BSA (0.1% wt/vol) and stored on ice until ready to use. BSA-coated and biocytin-coated particles were used interchangeably as controls. The final diameter of the virus-sized, CTB-coated particles was estimated to be 120 nm. This value was calculated knowing that the diameter of the carboxylate-modified latex beads was 93 nm and assuming a uniform coat of Neutravidin (7 nm) and CTB-biotin (6.5 nm), as described previously (10). Although the hydrodynamic diameters of RCA-I [relative molecular weight (Mr) 120,000] and Mal II (Mr 140,000) are undetermined, we expect that they are slightly greater than that of avidin (Mr 60,000).
Cell culture. The Caco-2BBe cell line was a generous gift from Dr. Mark Mooseker (Yale University). Caco-2 cells were grown in high-glucose DMEM (GIBCO BRL, Rockville, MD) supplemented with 10% FCS (HyClone Labs, Logan, UT), penicillin (100 IU/ml), streptomycin (100 µg/ml), and glutamine (2 µg/ml) at 37°C with an atmosphere containing 5% CO2, as described previously (41). For particle adhesion assays, cells were seeded at 2 × 105 cells/ml onto acetone-washed, 12-mm-diameter glass coverslips (Bellco Glass, Vineland, NJ) in 12-well plates (Costar, Cambridge, MA), as done previously (10). Seven days after confluence, cells were washed three times with PBS and then overlaid with soluble, biotinylated lectins (100 µg/ml) or particle mixtures and incubated with gentle rocking for 1 h at room temperature. Cells on coverslips were washed three times with PBS, fixed by immersion in formaldehyde (4% vol/vol in PBS), then inverted and mounted using Mowiol {0.5 g/ml glycerol, 0.1 g/ml Mowiol 4-88 (Calbiochem, San Diego, CA), 10 mg/ml diazabicylo[2.2.2]octane (Sigma) in 0.1 M Tris · HCl (pH 8.5)} onto Fisher Superfrost microscope slides (Fisher Scientific, Pittsburgh, PA) and viewed by fluorescence or phase-contrast microscopy.
Application of ligand-coated particles to live or fixed rabbit Peyer's patch mucosa. New Zealand White female rabbits weighing ~1.5 kg each were purchased from Charles River Laboratories (Wilmington, MA) and maintained in the animal resource facility at the Children's Hospital. All animal procedures were conducted in strict compliance with Guidelines for Animal Experimentation established by Harvard Medical School, the Children's Hospital, and the National Institutes of Health. Animals were fasted overnight with water ad libitum before surgery. To obtain Peyer's patches, rabbits were sedated with an intramuscular injection of 0.5 ml acetopromazine maleate (10 mg/ml) followed 45 min later by an intramuscular injection of ketamine (80 mg/kg) and xylazine (20 mg/kg). Animals were laparotomized and jejunoileal Peyer's patches were excised and treated as described below. Rabbits were euthanized by a single intravenous injection of pentobarbital sodium (100 mg/kg) into the marginal ear vein.
Freshly excised Peyer's patches were washed in PBS (25°C), and the luminal surfaces were blotted lightly with Whatman paper (3 MM) to remove excess mucus. Peyer's patches were then pinned to surgical cork board, and the mucosa was removed from the muscularis externa using a single-edged razor blade. For experiments using live tissue, the mucosae were cut into 2 × 2 mm pieces and each was placed in a single well of a 12-well tissue culture plate (Costar), which had been previously treated with RPMI 1640 (GIBCO BRL) and BSA (0.5% wt/vol). Taking special care not to damage the epithelium, the mucosa was washed with RPMI 1640 and overlaid with control and experimental particles (4 × 1010 total particles in 0.5 ml) or soluble, biotinylated lectins (100 µg/ml) and incubated at room temperature for 40 min. The tissue was carefully rinsed three times with PBS and either fixed for cryosectioning by immersion in formaldehyde (4% vol/vol) in PBS or for plastic embedding by immersion in formaldehyde (4% vol/vol) in 0.1 M cacodylate buffer (pH 7.2) (17). Lectin-treated tissue destined for cryosectioning was washed with PBS, incubated with glycine (0.1 M in PBS) to quench residual reactive aldehydes, blocked in PBS containing BSA (0.5% wt/vol) for 1 h, and labeled with fluorophore-conjugated streptavidin (50 µg/ml). For experiments using prefixed tissue, freshly excised mucosae were immersed in four volumes of formaldehyde (4% vol/vol) in PBS for 12 h. The tissue samples were then cut into 2 × 2 mm blocks, washed with PBS, incubated in 0.1 M glycine in PBS to quench residual reactive aldehydes, and blocked in PBS containing BSA (0.5% wt/vol) for 1 h. Blocks of tissue were then overlaid with lectins or particles for 40 min, rinsed, fixed again, and processed as described above for live tissue.Tissue sectioning, microscopy, and quantitation of particle
binding.
For cryosectioning, tissue samples were incubated for 1 h in sucrose
(15% wt/vol in PBS), followed by 10 min in Tissue-Tek optimum cutting
temperature embedding medium (Sakura Finetek, Torrance, CA), frozen in
liquid nitrogen-cooled isopentane, and stored at 20°C.
Frozen tissue sections were cut at 8-10 µm using a Leica
cryostat model CM3050 (Nussloch, Germany). Sections were captured on
Fisher Superfrost microscope slides and mounted with coverslips using
Mowiol. Slides were viewed under oil immersion using a Zeiss Axiophot
microscope (Carl Zeiss, Thornwood, NY) equipped for epifluorescence and
photographed using a 35 mm camera. Because protein-coated polystyrene
particles have a variable degree of nonspecific binding to intestinal
mucosa, 1:1 mixtures of ligand-coated and control particles were
applied in all experiments. For each experimental condition we analyzed
40 tissue sections, determining the numbers of ligand-coated particles
(fluorescent green) and control particles (fluorescent red) bound to
villus-associated epithelium and FAE. In specimens in which specific
binding occurred, there were on average >5,000 total particles bound
to 40 sections. For each tissue section we divided the number of
ligand-coated particles by the number of control particles to obtain a
relative index of binding. Binding indices were then averaged. All
experiments were done at least twice using two independently prepared
batches of ligand and ligand-coated particles and two different rabbits.
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RESULTS |
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CTB-coated, 120-nm particles did not adhere to live Peyer's patch
mucosa but adhered selectively to M cells on aldehyde-fixed tissues.
To probe glycolipids on the apical surfaces of intestinal epithelial
cells, we applied soluble, biotinylated CTB to rabbit Peyer's patch
mucosa. As observed previously (10), we found that CTB bound to GM1 in
apical plasma membranes of all intestinal epithelial cells on live and
formaldehyde-fixed Peyer's patch mucosa (Fig.
1A). Biotinylated BSA applied at
identical concentrations to Peyer's patch mucosa did not label
epithelial cells (Fig. 1B). To test whether virus-sized
particles have access to GM1, we applied CTB-coated 120-nm particles
(fluorescent green) mixed 1:1 with biocytin-coated control particles
(fluorescent red) to live Peyer's patch explants. We observed that
very few particles bound to either villus or FAE and that equal numbers
of CTB-coated and control particles were associated with the mucosal
surface (Table 1). Thus the FBBG on villus
and FAE enterocytes as well as the thin glycoprotein coat on M cells
were sufficient to prevent these virus-sized particles from gaining
access to glycolipid receptors.
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RCA-I binding sites on intestinal epithelial cells were inaccessible
to 120-nm particles.
Galactose-containing glycoproteins are proposed to be important
attachment sites for enteric bacteria. To confirm the distribution of
carbohydrates containing terminal (1,4)-linked galactose, we applied
soluble, biotinylated RCA-I to live or fixed Peyer's patch mucosa.
RCA-I labeled the surfaces of all epithelial cells strongly and
uniformly on live (Fig. 2, A and
B) and fixed tissue (data not shown). To test whether the
galactose epitopes recognized by RCA-I are accessible to virus-sized
particles in vivo, we applied a 1:1 mixture of RCA-I-coated 120-nm
particles (fluorescent green) and biocytin-quenched control particles
(fluorescent red) to live rabbit Peyer's patch mucosa. The RCA-I
particles did not show specific binding to any epithelial cells (Table
1). Both RCA-I and control particles adhered sparsely (Fig. 2C)
and RCA-I-coated particles did not bind to the intestinal epithelium
any better than control particles. On the FAE, nonspecific particle
binding occurred primarily on the apical surfaces of M cells. This was not surprising since rabbit Peyer's patch M cells have been shown previously to actively phagocytose uncoated polystyrene particles (10,
37). Aldehyde fixation did not enhance the availability of terminal
(1, 4) galactose sites to RCA-I-coated particles (data not shown).
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Mal II-coated 120-nm particles preferentially adhered to the FAE.
We next examined the accessibility of carbohydrate structures
containing (2,3)-linked sialic acid to virus-sized particles. On
live and fixed tissue, soluble, biotinylated Mal II adhered to the
luminal surfaces of villus enterocytes, FAE, and M cells (Fig.
3). On the FAE, Mal II labeled the apical
surfaces of enterocytes somewhat more intensely than the apical
surfaces of M cells, possibly reflecting an abundance of lectin-binding
sites in the thick FBBG (data not shown). To examine to what degree the
Mal II epitopes are accessible to 120-nm particles, we applied a 1:1
mixture of Mal II-coated particles (fluorescent green) and
biocytin-quenched control particles (fluorescent red) to live rabbit
Peyer's patch explants. On villus epithelium, Mal II-coated particles
bound in very low numbers and the ratio of Mal II to control particles was ~1:1 (Table 1). However, on the FAE Mal II particles adhered 4.5 times better than control particles (Fig.
4A and Table 1). Fluorescence
microscopy of plastic-embedded thin sections confirmed that Mal II
particles bound to both FAE and M cells (data not shown). These data
suggest that although glycoconjugates recognized by Mal II epitopes are
present on the apical surfaces of all intestinal epithelial cells in
vivo, they are only accessible to virus-sized particles on the FAE.
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Accessibility of oligosaccharides to bacterium-sized (1-µm) particles. In vitro binding assays have revealed bacterial adhesins that recognize a diversity of oligosaccharides and glycolipids (1, 19, 22, 43). Adhesins are often located on the tips of fimbriae and pili that extend hundreds of nanometers from the bacterial surface, suggesting that they may be able to penetrate the surface coats of host cells. This could be particularly important in the intestine, where the thick FBBG of enterocytes faces an environment rich in microorganisms. On this basis one might predict that many of the epithelial glycolipid and oligosaccharide structures that are potential microbial binding sites might be inaccessible to lectins or toxins immobilized on the surfaces of inert bacterium-sized particles that lack appendages. Indeed, we previously showed that ganglioside GM1 on live rabbit Peyer's patch mucosa is not accessible to CTB-coated, 1-µm beads (10).
To test the accessibility of oligosaccharides containing terminal ![]() |
DISCUSSION |
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For the majority of enteric pathogens, the primary event in mucosal infection is attachment to glycoconjugates on apical surfaces of intestinal epithelial cells. Indeed, bacteria and viruses express lectin-like adhesins on their surfaces (1, 19, 22, 27), and intestinal absorptive cells are coated with a heterogeneous array of glycolipids and protein-linked oligosaccharides (30, 45). The fact that glycoconjugates may extend from a few nanometers to hundreds of nanometers from the plasma membrane lead to the proposal that potential glycoconjugate binding sites may not be equally accessible to pathogens present in the intestinal lumen (25). In this study we have shown that certain glycolipid and protein-linked oligosaccharide epitopes that are present on the surfaces of all villus and FAE cells are not uniformly available for binding of ligands immobilized on virus- and bacterium-sized particles. The regional and cellular differences in receptor availability that we observed may explain in part the selective attachment of pathogenic viruses and bacteria to specific cell types in the intestinal mucosa.
Membrane glycolipids such as GM1, whose carbohydrate head extends only 2.5 nm from the membrane surface (47), should be relatively inaccessible to particles and most readily masked by membrane-associated glycoproteins. Since the thick FBBG on the microvilli of villus and FAE enterocytes had previously been shown to exclude CTB-coated particles as small as 28 nm in diameter, it was not surprising that 120-nm particles were also prevented from contacting enterocyte GM1. In contrast, the apical glycoprotein coat on rabbit Peyer's patch M cells is a relatively thin, irregular layer that extends only 20-30 nm from the membrane and allows binding of CTB-coated 28-nm particles to GM1 (10). This observation had raised hopes that virus-sized or even larger particles coated with CTB could be used to target vaccines to M cells and enhance the efficiency of mucosal immunization (16, 24). Unexpectedly, we found that the glycoprotein coat on live M cells was sufficient to prevent 120-nm particles from gaining access to GM1. This implies that CTB may not be useful for M cell targeting of particulate vaccine carriers such as copolymer microspheres but may be most effective as a component of macromolecular antigen complexes, as has been previously observed (31).
Our results also have implications for understanding rectal transmission of HIV-1. It was observed that cell-free HIV-1 adheres to, and is endocytosed by, M cells in mucosal explants from experimental animals (2) and humans (P. D. Smith, personal communication). Although it has been proposed that HIV-1 initially adheres to the glycosphingolipid galactosylceramide (9, 12), our data would suggest that it is unlikely that this molecule would be accessible to virus particles. Rather, we propose that the initial attachment of HIV-1 to the apical surfaces of M cells may involve an interaction of gp120 with more accessible membrane surface components (4, 32) or the interaction of host-derived adhesion molecules in the viral envelope with relevant ligands on M cells (21).
Mucinlike glycoproteins, including those that comprise the FBBG on the
apical surfaces of intestinal epithelial cells, contain abundant
heterogeneous oligosaccharide side chains that tend to form thick,
negatively charged gels (48). We hypothesized that the accessibility of
certain carbohydrate determinants in the enterocyte glycocalyx to
lectins immobilized on virus- or bacterium-sized particles may vary,
depending on their relative position within the FBBG. We found that
soluble RCA-I, which preferentially recognizes oligosaccharides
containing terminal (1,4)-linked galactose, adhered to rabbit villus
epithelium and FAE, but RCA-I immobilized on 120-nm or 1-µm particles
did not. The particles did bind specifically to our Caco-2 cells that
have abundant terminal galactose moieties and that lack a thick FBBG
(10). Although the exact topological locations of the epitopes
recognized by RCA-I on rabbit enterocytes and M cells are unknown, our
results suggest that they are sequestered within the FBBG or perhaps
located on smaller brush-border membrane glycoproteins.
Since oligosaccharides are commonly terminated by sialic acid, we
expected that glycoconjugates containing (2,3)-linked sialic acid
recognized by Mal II on both FAE and villus epithelium would be readily
available to lectin immobilized on particles. Surprisingly, on live
tissue 120-nm particles had access to Mal II epitopes only on the FAE
and not on villus epithelium. Access to the Mal II epitopes on the FAE
was size restricted, since 1-µm particles were unable to bind. Both
120-nm and 1-µm Mal II-coated particles adhered to aldehyde-fixed
tissue, indicating that the failure of the particles to bind to villus
epithelium was due to limited accessibility and not to a lack of Mal II
binding sites. These data add to the list of phenotypic differences
that are known to exist between the FAE and villus epithelium. For
example, all cells of the FAE lack expression of polymeric
immunoglobulin receptors (38) and show reduced brush border hydrolase
activity compared with villus enterocytes (44). Lectin and
antibody-binding studies in humans, mice, and rabbits revealed that FAE
may differ from villus epithelium in glycosylation patterns as well
(18, 46). Together, these data suggest that differences in both
receptor availability and receptor accessibility could enable pathogens to discriminate FAE from villus epithelium.
Viruses may overcome the glycocalyx barriers on enterocytes and M cells by at least two mechanisms. Karlsson et al. (26) proposed that an initial low-affinity, high-avidity interaction between viral surface protein(s) and readily accessible oligosaccharide epitopes in the glycocalyx might allow a virus to adhere to an epithelial cell and then progressively gain closer proximity to receptors in the host cell plasma membrane. Such a strategy might be used by rotavirus, which somehow penetrates the FBBG and infects villus epithelial cells. Alternatively, viruses could physically penetrate glycoprotein coats that are relatively thin, such as the 20- to 30-nm apical surface coat on M cells. This is suggested by the observation that extension of the reovirus outer capsid protein sigma 1, which forms a 45-nm-long fiber, is a prerequisite for selective adhesion to mouse Peyer's patch M cells (3, 36).
Despite the fact that both terminal galactose- and sialic
acid-containing glycoconjugates are recognized by enteric pathogens in
vitro, we found that these epitopes were inaccessible to lectin-coated, bacterium-sized particles in vivo. These results underscore the importance of the active strategies that bacteria have developed to
overcome the problem of limited receptor accessibility (1, 22, 25). For
example, Shigella flexneri secrete enzymes like -galactosidase that effectively degrade mucinlike glycoproteins (7,
42), and enterotoxigenic Escherichia coli express fimbriae that
could "reach" into the glycocalyx to attach to glycoconjugates containing
1-linked galactosyl residues (40). Although it has been
shown previously that tissue fixation can alter accessibility of
carbohydrate antigens to soluble lectins (20), our data demonstrate for
the first time that aldehyde fixation may enhance accessibility of
certain oligosaccharides to lectin-coated, bacterium-sized particles by
more than 20-fold. In light of these findings, bacterial adherence
studies on isolated glycoconjugates, tissue sections, or fixed tissue
should be interpreted with caution (8). In conclusion, more detailed
information about the accessibility of glycolipid and oligosaccharide
epitopes present on specific intestinal epithelial cells could lead to
a better understanding of mucosal infection and more effective antigen
delivery strategies.
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ACKNOWLEDGEMENTS |
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We thank Drs. Paul Giannasca, Karen Giannasca, and Larry Silbart for their helpful comments.
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FOOTNOTES |
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N. J. Mantis gratefully acknowledges the receipt of a postdoctoral fellowship from the Harvard AIDS Institute, Cambridge, MA [National Institutes of Health (NIH) Grant T32-A107387-07] and a NIH Individual National Research Service Award (NIH F32-AI10009-02). A. Frey was supported by a personal grant from the Bundesministerium fur Bildung, Wissenschaft, Forschung und Technologie, and research grant FR958/2-1 from the Deutsche Forschungsgemeinschaft. M. R. Neutra is supported by NIH research grants (HD-17557 and AI-34757) and by a NIH Center grant to the Harvard Digestive Diseases Center (DK-34854).
Present address for A. Frey: Center for Molecular Biology of Inflammation, University of Muenster, Germany.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: M. R. Neutra, GI Cell Biology Laboratory, Enders 1220, Children's Hospital, 300 Longwood Ave., Boston, MA 02115 (E-mail: neutra_m{at}a1.tch.harvard.edu).
Received 24 June 1999; accepted in final form 14 January 2000.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Abraham, AN,
Sharon N,
and
Ofek I.
Adhesion of bacteria to mucosal surfaces.
In: Mucosal Immunology (2nd ed.), edited by Ogra P,
Mestecky J,
Lamm ME,
Strober W,
Bienenstock J,
and McGhee JR.. San Diego: Academic, 1999, p. 31-42.
2.
Amerongen, HM,
Weltzin R,
Farnet CM,
Michetti P,
Haseltine WA,
and
Neutra MR.
Transepithelial transport of HIV-1 by intestinal M cells: a mechanism for transmission of AIDS.
J Acquir Immune Defic Syndr Hum Retrovirol
4:
760-765,
1991[ISI].
3.
Amerongen, HM,
Wilson GAR,
Fields BN,
and
Neutra MR.
Proteolytic processing of reovirus is required for adherence to intestinal M cells.
J Virol
68:
8428-8432,
1994[Abstract].
4.
Batinic, D,
and
Robey FA.
The V3 region of the envelope glycoprotein of human immunodeficiency virus type 1 binds sulfated polysaccharides and CD4-derived synthetic peptides.
J Biol Chem
267:
6664-6671,
1992
5.
Clark, MA,
Jepson MA,
Simmons NL,
and
Hirst BH.
Differential expression of lectin-binding sites defines mouse intestinal M cells.
J Histochem Cytochem
41:
1679-1687,
1993
6.
Compans, RW,
and
Herrler G.
Virus infection of epithelial cells.
In: Mucosal Immunology (2nd ed.), edited by Ogra P,
Mestecky J,
Lamm ME,
Strober W,
Bienenstock J,
and McGhee JR.. San Diego: Academic, 1999, p. 671-683.
7.
Corfield, AP,
Wagner SA,
Clamp JR,
Kriaris M,
and
Hoskins LC.
Mucin degradation in the human colon: production of sialidase, sialate O-acetyltransferase, N-acetylneuraminate lyase, arylesterase, and glycosulfatase activities by strains of fecal bacteria.
Infect Immun
60:
3971-3978,
1992[Abstract].
8.
Falk, P,
Boren T,
Haslam D,
and
Capron M.
Bacterial adhesion and colonization assays.
Methods Cell Biol
45:
165-192,
1994[ISI][Medline].
9.
Fantini, J,
Cook DG,
Nathanson N,
Spitalnik SL,
and
Gonzalez-Scarano F.
Infection of colonic epithelial cell lines by type 1 human immunodeficiency virus is associated with the cell surface expression of galactosylceramide, a potential alternative gp120 receptor.
Proc Natl Acad Sci USA
90:
2700-2704,
1993[Abstract].
10.
Frey, A,
Giannasca KT,
Weltsin R,
Giannasca PJ,
Reggio H,
Lencer WI,
and
Neutra MR.
Role of the glycocalyx in regulating access of microparticles to apical plasma membranes of intestinal epithelial cells: implications for microbial attachment and vaccine targeting.
J Exp Med
184:
1045-1059,
1996[Abstract].
11.
Fujimura, Y,
and
Owen RL.
M cells are portals of infections: clinical and pathophysiological aspects.
Infect Agents Dis
5:
144-156,
1996[ISI][Medline].
12.
Furuta, Y,
Eriksson K,
Svennerholm B,
Fredman P,
Horal P,
Jeansson S,
Vahlne A,
Holmgren J,
and
Czerkinsky C.
Infection of vaginal and colonic epithelial cells by the human immunodeficiency virus type 1 is neutralized by antibodies raised against conserved epitopes in the envelope glycoprotein.
Proc Natl Acad Sci USA
91:
12559-12563,
1994
13.
Gebert, A,
and
Hach G.
Differential binding of lectins to M cells and enterocytes in the rabbit cecum.
Gastroenterology
105:
1350-1361,
1993[ISI][Medline].
14.
Gentsch, JR,
and
Pacitti AF.
Effect of neuraminidase treatment of cells and effect of soluble glycoproteins on type 3 reovirus attachment to murine L cells.
J Virol
56:
356-364,
1985[ISI][Medline].
15.
Giannasca, KT,
Giannasca PJ,
and
Neutra MR.
Adherence of Salmonella typhimurium to Caco-2 cells: identification of a glycoconjugate receptor.
Infect Immun
64:
135-145,
1996[Abstract].
16.
Giannasca, P,
Boden J,
and
Monath T.
Targeted delivery of antigen to hamster nasal lymphoid tissue with M-cell-directed lectins.
Infect Immun
65:
4288-4298,
1997[Abstract].
17.
Giannasca, PJ,
Giannasca KT,
Falk P,
Gordon JI,
and
Neutra MR.
Regional differences in glycoconjugates of intestinal M cells in mice: potential targets for mucosal vaccines.
Am J Physiol Gastrointest Liver Physiol
267:
G1108-G1121,
1994
18.
Giannasca, PJ,
Giannasca KT,
Leichtner AM,
and
Neutra MR.
Human intestinal M cells display the sialyl Lewis A antigen.
Infect Immun
67:
946-953,
1999
19.
Goldhar, J.
Bacterial lectinlike adhesins: determination and specificity.
In: Bacterial Pathogenesis, edited by Clark VL,
and Bavoil PM.. San Diego: Academic, 1994, p. 211-230.
20.
Gonnella, PA,
and
Neutra MR.
Glycoconjugate distribution and mobility on apical membranes of absorptive cells of suckling rat ileum in vivo.
Anat Rec
213:
520-528,
1985[ISI][Medline].
21.
Hioe, CE,
Bastiani L,
Hildreth JE,
and
Zolla-Pazner S.
Role of cellular adhesion molecules in HIV type 1 infection and their impact on virus neutralization.
AIDS Res Hum Retroviruses
14, Suppl 3:
S247-254,
1998[ISI][Medline].
22.
Hultgren, SJ,
Abraham S,
Caparon M,
Falk P,
St. Geme JW,
and
Normark S.
Pilus and non-pilus bacterial adhesins: assembly and function in cell recognition.
Cell
73:
887-901,
1993[ISI][Medline].
23.
Ito, S.
Form and function of the glycocalyx on free cell surfaces.
Philos Trans R Soc Lond B Biol Sci
268:
55-66,
1974[ISI].
24.
Jepson, MA,
Clark MA,
Foster N,
Mason CM,
Bennett MK,
Simmons NL,
and
Hirst BH.
Targeting to intestinal M cells.
J Anat
189:
507-516,
1996[ISI][Medline].
25.
Karlsson, KA.
Meaning and therapeutic potential of microbial recognition of host glycoconjugates.
Mol Microbiol
29:
1-11,
1998[ISI][Medline].
26.
Karlsson, KA,
Angstrom J,
and
Teneberg S.
Characteristics of the recognition of host cell carbohydrates by viruses and bacteria.
In: Molecular Pathogenesis of Gastrointestinal Infections, edited by Wadstrom T.. New York: Plenum, 1991, p. 9-18.
27.
Lentz, TL.
The recognition event between virus and host cell receptor: a target for antiviral agents.
J Gen Virol
71:
751-766,
1990[ISI][Medline].
28.
Madara, JL.
Epithelia: biological principles of organization.
In: Textbook of Gastroenterology (2nd ed.), edited by Yamada T.. Philadelphia: Lippincott, 1995, p. 141-159.
29.
Maury, J,
Bernadac A,
Rigal A,
and
Maroux S.
Expression and glycosylation of the filamentous brush border glycocalyx (FBBG) during rabbit enterocyte differentiation along the crypt-villus axis.
J Cell Sci
108:
2705-2713,
1995
30.
Maury, J,
Nicoletti C,
Guzzo-Chambraud L,
and
Maroux S.
The filamentous brush border glycocalyx, a mucin-like marker of enterocyte hyper-polarization.
Eur J Biochem
228:
323-331,
1995[Abstract].
31.
McGhee, JR,
Mestecky J,
Dertzbaugh MT,
Eldridge JH,
Hirasawa M,
and
Kiyono H.
The mucosal immune system: from fundamental concepts to vaccine development.
Vaccine
10:
75-88,
1992[ISI][Medline].
32.
Mondor, I,
Ugolini S,
and
Sattentau QJ.
Human immunodeficiency virus type 1 attachment to HeLa CD4 cells is CD4 independent and gp120 dependent and requires cell surface heparans.
J Virol
72:
3623-3634,
1998
33.
Mooseker, MS.
Organization, chemistry, and assembly of the cytoskeletal apparatus of the intestinal brush border.
Annu Rev Cell Biol
1:
209-241,
1985[ISI].
34.
Neutra, MR,
Frey A,
and
Kraehenbuhl JP.
Epithelial M cells: gateways for mucosal infection and immunization.
Cell
86:
345-348,
1996[ISI][Medline].
35.
Neutra, MR,
Pringault E,
and
Kraehenbuhl JP.
Antigen sampling across epithelial barriers and induction of mucosal immune responses.
Annu Rev Immunol
14:
275-300,
1996[ISI][Medline].
36.
Nibert, M,
Furlong D,
and
Fields B.
Mechanisms of viral pathogenesis: distinct forms of reoviruses and their roles during replication in cells and host.
J Clin Invest
88:
727-734,
1991[ISI][Medline].
37.
Pappo, J,
and
Ermak TH.
Uptake and translocation of fluorescent latex particles by rabbit Peyer's patch follicle epithelium: a quantitative model for M cell uptake.
Clin Exp Immunol
76:
144-148,
1989[ISI][Medline].
38.
Pappo, J,
and
Owen RL.
Absence of secretory component expression by epithelial cells overlying rabbit gut-associated lymphoid tissue.
Gastroenterology
95:
1173-1174,
1988[ISI][Medline].
39.
Parashar, UD,
Breese JS,
Gentsch JR,
and
Glass RI.
Rotavirus.
Emerg Infect Dis
4:
561-570,
1998[ISI][Medline].
40.
Payne, D,
O'Reilly M,
and
Williamson D.
The K88 fimbrial adhesin of enterotoxigenic Escherichia coli binds 1-linked galactosyl residues in glycolipids.
Infect Immun
61:
3673-3677,
1993[Abstract].
41.
Peterson, MD,
and
Mooseker MS.
Characterization of the enterocyte-like brush border cytoskeleton of the Caco-2BBe clone of the human intestinal cell line Caco-2.
J Cell Sci
102:
581-600,
1992[Abstract].
42.
Prizont, R.
Degradation of intestinal glycoproteins by pathogenic Shigella flexneri.
Infect Immun
36:
615-620,
1982[ISI][Medline].
43.
Rafiee, P,
Leffler H,
Byrd JC,
Cassels FJ,
Boedeker EC,
and
Kim YS.
A sialoglycoprotein complex linked to the microvillus cytoskeleton acts as a receptor for pilus (AF/R1) mediated adhesion of enteropathogenic Escherichia coli (RDEC-1) in rabbit small intestine.
J Cell Biol
115:
1021-1029,
1991[Abstract].
44.
Savidge, T,
Smith M,
Mayel-Afshar S,
Collins A,
and
Freeman T.
Selective regulation of epithelial gene expression in rabbit Peyer's patch tissue.
Pflügers Arch
428:
391-399,
1994[ISI][Medline].
45.
Semenza, G.
Anchoring and biosynthesis of stalked brush border membrane proteins: glycosidases and peptidases of enterocytes and renal tubuli.
Annu Rev Cell Biol
2:
255-313,
1986[ISI].
46.
Sharma, R,
van Damme EJM,
Peumans WJ,
Sarsfield P,
and
Schumacher U.
Lectin binding reveals divergent carbohydrate expression in human and mouse Peyer's patches.
Histochem Cell Biol
105:
459-465,
1996[ISI][Medline].
47.
Spangler, BD.
Structure and function of cholera toxin and the related Escherichia coli heat labile enterotoxin.
Microbiol Rev
56:
622-647,
1992[Abstract].
48.
Van Klinken, BJ-W,
Dekker J,
Buller H,
and
Einerhand E.
Mucin gene structure and expression: protection vs. adhesion.
Am J Physiol Gastrointest Liver Physiol
269:
G613-G627,
1995
49.
Willoughby, RE.
Rotaviruses preferentially bind O-linked sialylglycoconjugates and sialomucins.
Glycobiology
3:
437-445,
1993[Abstract].
50.
Willoughby, RE,
Yolken RH,
and
Schnaar RL.
Rotaviruses specifically bind to the neutral glycosphingolipid asialo-GM1.
J Virol
64:
4830-4835,
1990[ISI][Medline].
51.
Wolf, J,
Rubin D,
Finberg R,
Kauffman R,
Sharpe A,
Trier J,
and
Fields B.
Intestinal M cells: a pathway for entry of reovirus into the host.
Science
212:
471-472,
1981[ISI][Medline].