Campylobacter jejuni Binds Intestinal H(O) Antigen (Fucalpha 1, 2Galbeta 1, 4GlcNAc), and Fucosyloligosaccharides of Human Milk Inhibit Its Binding and Infection*

Guillermo M. Ruiz-PalaciosDagger §, Luz Elena CervantesDagger , Pilar RamosDagger , Bibiana Chavez-Munguia, and David S. Newburg||

From the Dagger  Department of Infectious Diseases, National Institute of Medical Sciences and Nutrition, Vasco de Quiroga 15, Mexico D. F. 14000, Mexico, the  Department of Experimental Pathology, Centro de Investigacion y Estudios Avanzados, I. P. N., Av. Instituto Politecnico Nacional 2508, Mexico D. F. 07360, Mexico, and the || Program in Glycobiology, Shriver Center, University of Massachusetts Medical School, Waltham, Massachusetts 02452

Received for publication, July 31, 2002, and in revised form, January 31, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The most common cause of infant mortality is diarrhea; the most common cause of bacterial diarrhea is Campylobacter jejuni, which is also the primary cause of motor neuron paralysis. The first step in campylobacter pathogenesis is adherence to intestinal mucosa. We found that such binding was inhibited in vitro by human milk and, with high avidity, by alpha 1,2-fucosylated carbohydrate moieties containing the H(O) blood group epitope (Fucalpha 1,2Galbeta 1,4GlcNAc ... ). In studies on the mechanism of adherence, campylobacter, which normally does not bind to Chinese hamster ovary cells, bound avidly when the cells were transfected with a human alpha 1,2-fucosyltransferase gene that caused overexpression of H-2 antigen; binding was specifically inhibited by H-2 ligands (lectins Ulex europaeus and Lotus tetragonolobus and H-2 monoclonal antibody), H-2 mimetics, and human milk oligosaccharides. Human milk oligosaccharides inhibited campylobacter colonization of mice in vivo and human intestinal mucosa ex vivo. Campylobacter colonization of nursing mouse pups was inhibited if their dams had been transfected with a human alpha 1,2-fucosyltransferase gene that caused expression of H(O) antigen in milk. We conclude that campylobacter binding to intestinal H-2 antigen is essential for infection. Milk fucosyloligosaccharides and specific fucosyl alpha 1,2-linked molecules inhibit this binding and may represent a novel class of antimicrobial agents.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Intestinal infection by Campylobacter jejuni is one of the most common causes of diarrhea worldwide and is the primary cause of ascending motor neuron paralysis (Guillain-Barré syndrome and other variants) (1). This enteropathogen induces disease by a series of complex steps that include motility and tropism to the ileum and proximal colon (2-4), adherence to and invasion of intestinal epithelial cells (5-8), and production of enterotoxin (9) and cytotoxins (10, 11). Thus, cell adherence and invasion are essential for the pathogenesis of campylobacter diarrhea. However, binding of host cell surface components by campylobacter is not as well defined as binding by other bacterial enteropathogens such as shigella, yersinia, and salmonella species (12). Although some campylobacter surface proteins associated with binding have been characterized and cloned (13-15), their precise role has not been established (16). Furthermore, little is known about the host cell surface receptors for this organism.

Cohort studies of Mexican children indicate that breast feeding protects against campylobacter diarrhea; milk-specific antibodies contribute to this protection (17), but other anti-infective components also may be involved. Nonimmunoglobulin fractions of human milk protect against other respiratory and gastrointestinal tract infections (18, 19). This protective effect is attributed to blocking activities by complex carbohydrate molecules in human milk, including glycoconjugates and oligosaccharides, which are able to function as soluble receptor analogs (20).

At high concentrations, L-fucose inhibits adherence of campylobacter to epithelial cells in vitro (21), suggesting that cell receptors for campylobacter may be fucosylated carbohydrate epitopes. Fucosylated oligosaccharide moieties that constitute some of the blood group antigens on erythrocytes are also present in gut epithelial cells and human milk (22). Recently, these antigens have been shown to act as receptors for some enteropathogens, e.g. Lewisb (Leb) for Helicobacter pylori (23-25) and H-1 epitopes for calicivirus (26). Therefore, we hypothesized that the host cell receptor for campylobacter might contain a fucosylated epitope of a blood group antigen.

This hypothesis was tested in three sets of experiments. The first set was conducted to determine whether the fucosylated oligosaccharide fraction of human milk inhibits campylobacter adherence to epithelial cells in vitro. The second set was conducted to clarify the nature of the host cell receptors involved. The third set of experiments tested biological significance: whether fucosylated human milk oligosaccharides inhibit campylobacter colonization of human intestinal mucosa ex vivo and mouse intestine in vivo, and whether the presence of 2-linked fucosylglycoconjugates in milk of mice transfected with pWAP alpha 1,2-FUT1 inhibits campylobacter colonization in the intestine of their nursing pups. pWAP alpha 1,2-FUT1 expresses H (O) antigen mainly in mammary gland during lactation.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Milk Fractions-- Milk lipids were prepared by chloroform/methanol (2:1) extraction of lyophilized milk pooled from 40 mothers. The protein fraction was prepared from similar pooled milk that was defatted by centrifugation (4,000 × g, 30 min, 4 °C), concentrated on a 10,000 MWCO ultrafilter, and dialyzed; antibodies were removed by passing this fraction through a protein A affinity column followed by passage through an Artocarpus integrifolia (jacalin) lectin affinity column. Oligosaccharide fractions were obtained from pooled human milk (New England Regional Milk Bank, Worcester, MA) as previously described (27). The cream was removed by centrifugation; skimmed milk was filtered through glass wool and mixed with ice-cold acetone to precipitate the proteins, stirred overnight at 4 °C, and centrifuged. The supernatant was passed through a charcoal column, and the resulting crude oligosaccharide fraction was passed through an ion exchange column, yielding neutral and acidic (anionic) oligosaccharides. The neutral fraction (25 mg) was dissolved in buffered aqueous bicarbonate (25 mM) in 150 mM NaCl, pH 7.0, containing 0.01% NaN3, applied to a 50-ml Ulex europaeus lectin affinity (UEA I)1 column, and eluted with 500 ml of the same buffer. The bound oligosaccharide fraction was eluted with 500 ml of an aqueous solution of 1 M NaCl buffered with 25 mM NaOAc, pH 4.0, containing 0.01% NaN3. To isolate oligosaccharides from their elution buffers, the lectin column eluates were passed through a charcoal column, rinsed with water, and eluted with 65% ethanolaq. The oligosaccharides were quantified by gravimetry, and the ratios of their monosaccharides were determined by gas chromatography of the trimethylsilyl derivatives of their hydrolyzed monosaccharides. The UEA I-adherent fraction, about 10% of the neutral oligosaccharide fraction, contained a significantly higher ratio of fucose than the nonadherent fraction. Thin-layer chromatography indicated that each of these two fractions contained distinct oligosaccharide components. All fractions were tested for their ability to inhibit association of campylobacter with carcinoma-derived human epithelial (HEp-2) cells.

C. jejuni Strains-- Prototype invasive strains 84sp, 135ip, 166ip, 173ip, 180ip, 187ip, 193ip, 225sp, 268ip, 287ip, and 383ip were isolated from children with inflammatory diarrhea; the prototype adherent strain 10sp was isolated from a child with noninflammatory diarrhea; the nonadherent strains 17sp, 49sp, 50sp, and 57sp were from healthy children (28).

HEp-2 Cell Adherence Assay for Campylobacter-- Campylobacter binding to a monolayer of HEp-2 cells was studied as previously described (29, 30). Briefly, HEp-2 cells were grown to confluency in minimum essential medium (MEM) supplemented with 5% fetal bovine serum, Earls salts, and L-glutamine at 37 °C in 5% CO2; cells were released with trypsin, and a cell suspension of 2 × 105 was transferred into multichambered culture slides (Falcon, Franklin Lakes, NJ). For inhibition assays, 100-µl suspensions of 9 × 108 bacteria of C. jejuni strains previously incubated with lactose (5 mg/ml), lipid (1 mg/ml), nonimmunoglobulin protein (1 mg/ml), and oligosaccharide milk fractions (3 mg/ml), were added to monolayers of HEp-2 cells in 8-chamber tissue culture slides, incubated at 37 °C for 5 h, washed, and stained by the Warthin-Starry method. To calculate the cell association index, 100 cells were counted in each well, and the index was the number of cells per well that were infected by at least 10 campylobacter organisms per cell. A strain was considered pathogenic (invasive) when the association index was >= 20%. Results of triplicate assays were given as the percent inhibition of campylobacter cell association relative to identical positive controls to which no milk fractions were added. Inhibition of cell association was considered significant when there was >= 20% inhibition. For the assays of cell association inhibition by milk fractions, pathogenic strains 10sp, 84sp, 166ip, 287ip, and 383ip, and non-pathogenic strains 50sp and 57sp were tested.

Bacterial Binding Blotting Assays-- The ability of campylobacter to bind to immobilized glycoconjugates on nitrocellulose membranes was assayed with digoxigenin (DIG)-labeled bacteria (23, 24). Blood group antigens, linked to neoglycoproteins, were applied individually to lanes for SDS-PAGE at equimolar concentrations of 6.3 × 10-10 M blood group oligosaccharide per lane: Leb (2.02 µg, 32 mol of sugar/mol of protein), H-1 (2.3 µg, 25 mol of sugar/mol of protein), H-2 (2.0 µg, 26 mol of sugar/mol of protein), Tri-Lex (2.84 µg, 23 mol of sugar/mol of protein), Ley (2.1 µg, 26 mol of sugar/mol of protein), Lea (2.44 µg, 23 mol of sugar/mol of protein), Lex (2.71 µg, 20 mol of sugar/mol of protein), A (3.16 µg, 18 mol of sugar/mol of protein) and B (2.57 µg, 19 mol of sugar/mol of protein) (Iso Sep AB, Tullingen, Sweden). These neoglycoproteins were transferred from the gel to nitrocellulose membranes. Membrane was blocked with BB2 (consisting of Tris-buffered saline (TBS), pH 7.5, 1% blocking reagent (Roche Molecular Biochemicals, Basel, Switzerland), 1 mM MnCl2, 1 mM MgCl2, and 1 mM CaCl2) to minimize nonspecific binding. The membrane was washed twice in TBS, and a 10-ml suspension of 0.2 OD at 600 nm of DIG-labeled invasive C. jejuni was added and incubated for 4 h at room temperature. The membrane was washed thoroughly six times, incubated with anti-DIG alkaline phosphatase conjugate for 1 h at room temperature, washed five times, and stained with 5-bromo-4-chloro-3-indolyl phosphate (X-phosphate) and Tris-buffered nitroblue tetrazolium substrate in saline (pH 9.5) (Roche Molecular Biochemicals).

Specificity of campylobacter binding to blood group antigens was determined by transferring neoglycoproteins to nitrocellulose membranes preincubated with monoclonal antibodies (mAbs) to antigens H-1, Lea, Lex, Leb (Signet Laboratories, Dedham, MA), H-2, or Ley (Accurate Chemical, Westbury, NY) at dilutions of 1:10 and 1:20. A 100-µl suspension of 0.2 OD at 600 nm of DIG-labeled invasive C. jejuni was added and incubated for 4 h at room temperature and the membranes were washed, incubated with anti-DIG-alkaline phosphatase, washed, and stained with X-phosphate and nitroblue tetrazolium.

Specificity of campylobacter binding to H-1 and H-2 blood group antigens was determined also by inhibition with 2'-fucosyllactose in a Western blot assay. Briefly, equimolar concentrations of neoglycoproteins H-1 and H-2 were run in SDS-PAGE, transferred to nitrocellulose, blocked, and incubated with a DIG-labeled C. jejuni suspension of 0.2 OD at 600 nm preincubated with different concentrations of 2'-fucosyllactose (1, 10, 20, 50, and 250 µg per ml). After 4 h of incubation at room temperature with gentle agitation, the membranes were washed six times, incubated with anti-DIG alkaline phosphatase, washed, and stained as above.

To determine the relative affinity of UEA I lectin to blood group antigens, neoglycoproteins were transferred to nitrocellulose membranes, which were then incubated with BB2 buffer overnight and washed twice in TBS. Membranes were incubated in peroxidase-labeled UEA I (5 µg/ml) for 4 h, washed six times for 5 min each in TBS, and stained with alpha -naphthol/H2O2/TBS (15 ml of TBS, 3 ml of MeOH, 9 mg of alpha -naphthol, 9 µl of H2O2).

Twelve pathogenic and four non-pathogenic strains were tested; for each strain, assays were performed on different days and at least three times. Differences in binding by pathogenic versus nonpathogenic strains of campylobacter to alpha 1,2 moieties were analyzed for statistical significance by Fisher's Exact Ratios.

Chinese Hamster Ovary (CHO) Cells Transfected with Human Glycosyltransferases-- CHO cells transfected with alpha 1,2-fucosyltransferase (CHO-FUT1), alpha 1,3/4-fucoslytransferase (CHO-FUT3), and alpha 1,3-fucosyltransferase (CHO-FUT4), and parental CHO cells transfected with only the vector pCDM7 (CHO-V) (31), were used to test bacterial binding and bacteria-induced host cell agglutination. Each line was grown under the conditions recommended: CHO-FUT1 cells were grown to confluency in Dulbecco's modified Eagle's medium (high glucose) containing additions of 10% fetal bovine serum, 110 µg/ml sodium pyruvate, 50 µg/ml gentamicin sulfate, 15 µg/ml hypoxanthine, 1 µg/ml aminopterine, and 5.15 µg/ml thymidine. CHO-FUT3 and CHO-FUT4 cells were grown in MEM without phenol red containing additions of 10% fetal bovine serum, 10 µg/ml L-glutamine, 10 µg/ml ribonucleosides, 10 µg/ml desoxyribonucleosides, and 400 µg/ml geneticin. Parental CHO-V cells were grown in Iscoves modified Dulbecco's medium containing additions of 10% fetal bovine serum, 5 µg/ml penicillin-streptomycin, 10 mM hypoxanthine, and 1.6 mM thymidine.

Immunofluorescence Assays-- CHO-FUT1, CHO-FUT3, CHO-FUT4, and parental CHO-V cells with vector pCDM7 alone were grown as described above in 8-chamber slides for 18 h. The monolayer cells were washed three times with PBS, pH 7.3 and fixed at room temperature with formalin for 1 h. Cells were washed and treated with 1% BSA in PBS for 30 min to minimize nonspecific binding. The monolayer was then incubated at room temperature for 90 min with mouse mAbs to H-2, Lex, Ley, H-1, Lea, and Leb diluted 1:30 in PBS with 0.1% BSA, washed three times with PBS, and treated with a rabbit polyclonal anti-mouse antibody conjugated with flourescein isothiocyanate (FITC) diluted 1:40 in PBS with 0.1% BSA. The cells were washed three times with PBS, counterstained with Evans blue, and observed by immunofluorescence microscopy.

Campylobacter Binding to Monolayers of Transfected CHO Cells-- The ability of campylobacter to bind CHO cells that had been transfected with FUT1, the human gene for alpha 1,2-fucosyltransferase needed for the synthesis of the alpha 1,2-fucose-containing H-antigen, was assessed. Controls included parental CHO-V cells. Cells were grown to confluency (31), and monolayers were harvested, seeded into each well of an 8-chamber slide, incubated for 18 h, washed, and incubated with a suspension of 9 × 108 bacteria/ml. Wells were rinsed six times with PBS, fixed with 10% formalin for 1 h, stained by the Warthin-Starry method, and examined under oil immersion with light microscopy. Identical preparations grown on round cover slips were prepared for scanning electron microscopy by fixing in 2% glutaraldehyde, dehydration through a graded series of solvents, and surface gold deposition.

Inhibition of Binding-- alpha 1,2-Fucosyl ligands and homologs were tested for their ability to inhibit binding of C. jejuni strains 84sp and 287ip to CHO-FUT1 cells. Inhibition by molecules that bind to H-2 ligand (i.e. anti-H-2 mAb at 1:10, 1:20, and 1:40 dilutions and the lectins UEA I and Lotus tetragonolobus (TP) at 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, and 3.2 mg/ml) was measured on monolayers of CHO-FUT1 cells incubated in 8-well chamber slides for 1 h with each of the alpha 1,2-fucosyl ligands before addition of 100 µl of the bacterial suspension containing 1 × 108 bacteria/ml; as controls anti-H-1 and anti-A mAbs at the same dilutions, and the lectin Dolichus biflorus at the same concentrations were used. To test inhibition by homologs of cell surface receptors, 100 µl of the bacterial suspension was incubated with human milk neutral oligosaccharides (0.5, 1, 1.5, and 2.0 mg/ml), neoglycoprotein BSA-H-2 (0.5, 1, 1.5, and 2.0 mg/ml) (IsoSep AB), or 2'-fucosyllactose (0.02, 0.04, 0.08, 0.16, 0.625, 1.25, 2.5, and 5 mg/ml, equal to 0.04, 0.08, 0.16, 0.32, 1.28, 2.56, 5.12, and 10.2 µmol/ml) before being added to the cell monolayer; as controls lactose (1, 2, 4, 8, and 16 mg/ml, equal to 2.9, 5.8, 11.6, 23.3, 46.7 µmol/ml) and BSA (0.4, 0.8 and 1.6 mg/ml, equal to 6.1, 12.1, 24.2 nmol/ml) were used. For both assays, after a 3-h incubation at 37 °C, wells were rinsed six times with PBS, cells were lysed with 1% Triton X-100, and CFU of campylobacter per well were determined. Data are presented as percent inhibition of campylobacter association to cells relative to positive controls to which no alpha 1,2-fucosyl ligands or homologs were added.

Cell Agglutination-- To assay the binding of campylobacter to specific glycans expressed on the surface of glycosyltransferase-transfected CHO cells, campylobacter-induced agglutination of a suspension of detached CHO cells was measured. Confluent monolayers of CHO-FUT1, CHO-FUT3, CHO-FUT4, and parental CHO-V were detached with trypsin-EDTA and washed three times in Tris buffer (50 mM Tris, 1 mM CaCl2, 1 mM MgCl2, pH 8). Aliquots (10 µl) of a cell suspension (3 × 106 cells/ml) were placed on microscope slides, and an equal volume of UEA I solution (1 mg/ml) or a suspension of 108 campylobacter was added. The microscope slides were gently shaken, and agglutination was determined by a blinded observer using direct and microscopic observation 3 min after the addition of lectin or bacterial suspension. Strong agglutination was designated 3+, intermediate agglutination was 2+, perceptible but weak agglutination was 1+, and no agglutination was 0.

Colonization of Mouse Gut-- A mouse model for campylobacter colonization (32) was used to investigate inhibition of colonization by human milk oligosaccharides. Groups of 6 BALB/c mice weighing 10-20 g each and caged in pairs, received 2 mg of neutral milk oligosaccharides in 100 µl of PBS by oral intubation 2 h before, during, and 2 h after oral challenge with 104 or 108 CFU of C. jejuni 287ip (total of 6 mg of oligosaccharides, equivalent to the amount in 1 ml of human milk, over 4 h). Controls were given 100 µl of PBS before, during, and after challenge with the same bacterial suspensions. To test for any adverse effects due to the administration of oligosaccharides per se, another control group received only 2 mg of oligosaccharides; no effects were observed in these controls over the course of the experiment. Shedding of campylobacter was determined by daily quantitative stool cultures for 5 days after challenge. All animal protocols were reviewed and approved by the Animal Care Committee of the National Institute of Medical Sciences and Nutrition.

Human Intestinal Adherence-- Normal ileum specimens from patients who required intestinal resection were transported to the laboratory at 4 °C in PBS, pH 7.3, supplemented with aprotinin (2 µg/ml), and then washed several times with cold PBS. Mucus was removed carefully with soft tissue paper, and segments were cut into 1-cm2 pieces. To measure bacterial binding to human intestinal tissue, the pieces were incubated for 2 h at 37 °C under microaerophilic conditions in 1.5-2.0 ml of aprotinin (1 µg/ml) bacterial suspension (0.1 OD at 600 nm), which had been previously incubated with UEA I-affinity-purified oligosaccharide fraction (0.2 mg/ml, one-half its natural concentration in pooled human milk), 2'-fucosyllactose (2 mg/ml) or saline alone. Campylobacter strains tested were pathogenic C. jejuni 287ip and nonpathogenic 57sp. Samples were then washed six times in PBS, homogenized with 1 ml of isotonic saline solution, and centrifuged at 50 × g for 5 min. The supernatant was diluted 10- and 20-fold and plated. Bacterial binding was expressed as CFU per cm2 of tissue.

Expression of H-2 Epitope in Milk of Transgenic Mice-- BG/SJL mice transfected with a plasmid containing the human FUT1 gene, the poly(A) signal for bovine growth hormone, and a murine whey acidic protein promoter that directs expression primarily to lactating mammary gland (33) were also used in these experiments. PCR examination of each dam verified that they were transgenic. Each lactating dam was caged separately with her litter. Preliminary stool cultures from mothers and cultured intestinal homogenates from one pup from each litter demonstrated that they had not been previously infected with campylobacter. Suckling mice were then orally inoculated with 10 µl of 104, 108, or 109 CFU/ml (i.e. 102, 106, and 107 per animal) of invasive strain 287ip and returned to their dams to continue nursing. Control pups from nontransgenic dams were inoculated with 10 µl of 108 CFU/ml (106 per animal). To determine colonization, one mouse from each litter (at least 10 individual pups) was sacrificed every third day for 15 days. One centimeter of the distal ileum and the cecum were resected and placed separately in 1 ml of PBS, weighed, homogenized, and centrifuged at 1200 × g for 10 min. Serial dilutions were cultured to determine CFUs per gram of tissue.

The amount of colonization was expressed as the percentage of pups positive for campylobacter at each time period tested. Differences between the pups challenged with an innoculum of 106 campylobacter and nursing nontransgenic wild-type dams versus those nursing transgenic dams were evaluated by the Mann-Whitney test. Differences between all groups and across all times tested were evaluated by analysis of variance followed by estimation of the regression coefficients.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Effect of Human Milk Fractions on Campylobacter Adherence-- Protein, lipid, and carbohydrate fractions prepared from pooled human milk (27) were tested for their ability to inhibit campylobacter adherence to HEp-2 cells (29, 30). The total oligosaccharide fraction at 3 mg/ml, one-half of the mean concentration in pooled milk, inhibited infection of HEp-2 cells by any of the prototype invasive campylobacter strains (Fig. 1), regardless of the serotype. Such inhibition was not observed with the lactose, lipid, or nonimmunoglobulin protein fractions (Fig. 1). Furthermore, the active oligosaccharide fraction caused detectable inhibition at concentrations of only one-twentieth of its normal concentration in human milk (0.3 mg/ml, not shown).


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Fig. 1.   Inhibition of campylobacter binding to HEp-2 cells by fucosylated oligosaccharides of human milk. A monolayer of HEp-2 cells seeded in 8-chamber slides was infected with a suspension of 9 × 108 invasive C. jejuni strain 287ip. This strain abundantly infects more than 80% of each HEp-2 cell monolayer. Preincubation of bacteria with lactose (5 mg/ml), lipid (2 mg/ml), or nonimmunoglobulin protein (2 mg/ml) fractions of pooled human milk did not inhibit campylobacter attachment to HEp-2 cells, but preincubation with total oligosaccharides (3 mg/ml, half the concentration found in human milk) did. The inhibitory activity of the crude oligosaccharide fraction of milk remained in the neutral fraction obtained from an ion exchange column, and in the fucosylated oligosaccharide fraction that was retained by the U. europaeus (UEA I) lectin affinity column (mean ± S.E.; n = 3). Results of triplicate assays are given as percent inhibition of bacterial cell association relative to a control preincubated only with minimal essential medium without the milk fractions.

When the crude oligosaccharide fraction was further separated into neutral and acidic fractions, only the neutral fraction inhibited campylobacter binding (p < 0.005). After this fraction was passed through a UEA I-affinity column, the inhibitory activity was observed only in the fucosylated fraction retained by the column (p < 0.01; Fig. 1). Previous studies have demonstrated that UEA I lectin binds predominantly to alpha 1,2-linked fucose, with especially high avidity for the Fucalpha 1,2Galbeta 1,4GlcNAc ... moiety (34), which includes the H-2, Ley, and also binds to a lesser extent to the H-1, Leb, and Lea blood group antigens (35). The ability of UEA I-binding milk oligosaccharides to inhibit campylobacter adherence to HEp-2 cells implies that fucosylated oligosaccharides of human milk, possibly those containing H epitopes, might inhibit campylobacter adherence to its host cell receptor.

Campylobacter Binding to Blood Group Antigens-- To define the nature of the host cell glycoconjugate ligands for campylobacter, we measured the ability of the bacterium to bind to an array of fucosylated carbohydrate chains linked to albumin (neoglycoproteins) and immobilized on a nitrocellulose membrane. Bacterial binding varied according to the type of campylobacter isolate: an isolate from a patient with inflammatory diarrhea (strain 287ip), which had the ability to adhere to and invade HEp-2 cells, bound avidly to all fucosylated neoglycoproteins; however, the strongest binding was to H-1 and H-2 antigens (Fig. 2A). An isolate from an asymptomatic individual, strain 50sp, did not adhere to HEp-2 cells or to any of the neoglycoproteins tested (Fig. 2B). 10 of 12 pathogenic C. jejuni strains (84sp, 135ip, 166ip, 180ip, 187ip, 225sp, 268ip, 383ip, 10sp, and 287ip, included in Fig. 2A) bound to the fucosylated antigens by Western blot, whereas none of four nonpathogenic strains (17sp, 49sp, 50sp, and 57sp) bound (Fig. 2B; p = 0.01). Thus, the pathogenic potential of different strains of campylobacter seems to be associated with their ability to bind these H-Lewis blood group epitopes.


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Fig. 2.   DIG-labeled C. jejuni strains bind to immobilized blood group antigens in Western blots, indicating that campylobacter virulence is associated with H antigen binding. Panel A, binding of C. jejuni strains to tissue blood group antigens was assessed by a bacterial binding blotting assay. Equimolar concentrations of 6.3 × 10-10 M of tissue blood group antigen oligosaccharides were applied to each lane: Leb (2.0 µg, lane 2), H-1 (2.3 µg, lane 3), H-2 (2.0 µg, lane 4), tri-Lex (2.8 µg, lane 5), Ley (2.1 µg, lane 6), Lea (2.4 µg, lane 7), Lex (2.7 µg, lane 8), group A (3.2 µg, lane 9), or group B (2.6 µg, lane 10), and aliquots (2 µg) of bovine serum albumin alone (lane 1), were resolved on a 7.5% SDS-PAGE, transferred to nitrocellulose, and blotted with DIG-labeled campylobacter strains. C. jejuni strain 287ip, a pathogenic strain with a cell association index of 98%, bound to all tissue blood group antigen neoglycoproteins, but most strongly to H-1 and H-2. Panel B, 10 of 12 pathogenic strains of C. jejuni bound to H-Lewis epitope-containing neoglycoproteins (the nine shown in this figure and 287ip in Fig. 2A above), while none of the four non-pathogenic strains bound, suggesting that a common receptor for pathogenic, but not non-pathogenic, campylobacter may be H-Lewis epitopes.

The specificity of campylobacter binding to H epitopes was confirmed in Western blot competition assays that tested mAbs for their ability to specifically inhibit binding of the bacterium to the panel of blood group antigens. Strong inhibition of bacterial binding was observed with mAbs against the monofucosylated H-2 (Fig. 3A, lanes 4-6) and the analogous difucosylated Ley antigens (not shown), whereas no inhibition was observed with anti-H-1 mAb (Fig. 3A, lanes 1-3), and weak inhibition occurred with anti-Lea, -Leb, and -Lex (not shown).


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Fig. 3.   C. jejuni binding is specific to the H-2 epitope. Panel A, bacterial binding to H-1 (lane 1, C) is not inhibited by anti-H-1 antibody at dilutions of 1:10 (lane 2) or 1:20 (lane 3), indicating nonspecific binding. Bacterial binding to H-2 receptor (lane 4, C) is inhibited by monoclonal antibodies against H-2 at dilutions of 1:10 (lane 5) and 1:20 (lane 6), consistent with H-2 being the specific receptor. Panel B, bacterial binding to H-2 (lane 2) was inhibited by 2'-fucosyllactose at concentrations of 10 µg/ml (lane 6), 20 µg/ml (lane 8), 50 µg/ml (lane 10), and 250 µg/ml (lane 12). Bacterial binding to H-1 (lane 1) was only inhibited at 250 µg/ml (lane 11), suggesting that campylobacter binding to H-1 is nonspecific.

Specificity of campylobacter binding to H-1 and H-2 blood group antigens was confirmed by comparing the inhibition of binding using 2'-fucosyllactose, the shortest structure that defines the group of alpha 1,2-fucosylated residues. A strong inhibition of the binding of bacteria to H-2 was observed with concentrations as low as 10 µg/ml (Fig. 3B, lanes 6, 8, 10, 12, and 14), while a concentration 25 times higher (250 µg/ml) was necessary to inhibit the binding to H-1 (Fig. 3B, lanes 13 and 15). These results suggest that the fucosyl alpha 1,2 moiety is a critical feature for campylobacter binding, and that the H-2 epitope is the preferred fucosyl alpha 1,2 structure.

Specificity of H-2 Binding by Campylobacter in Transfected Cells-- To test the hypothesis that cellular expression of alpha 1,2-fucosyl moieties (H antigen) allows campylobacter binding, CHO cells were transfected with the FUT1 gene for human alpha 1,2-fucosyltransferase whose expression product catalyzes the final step in the synthesis of H antigen. Parental CHO cells do not express alpha 1,2-linked fucose-containing epitopes on their surface, and have low campylobacter binding affinity relative to HEp-2 cells. However, FUT1-transfected CHO cells express H(O) blood group antigens (31). Transfection with the alpha 1,2-fucosyltransferase human gene confers on CHO cells the ability to complete the synthesis of all fucosyl alpha 1,2-containing antigens; the actual products will depend upon the availability of endogenous substrates. Identical cells were likewise transfected with alpha 1,3/4-fucosyltransferase (the Lewis gene, FUT3), with human alpha 1,3-fucosyltransferase (FUT4), or with the vector pCDM7 alone. The cells transfected with FUT1 reacted with mAbs to H-2 antigen only, but not with mAbs to H-1, Lea, Leb, Lex, or Ley (Fig. 4A). Those transfected with FUT3 reacted with Ley only, while those transfected with FUT4 reacted with Lex only, and those cells carrying the vector only did not react with any of these H-Lewis antibodies (data not shown). Thus, the transfected cells expressed the expected fucose-linked carbohydrate structures. Markedly greater numbers of campylobacter bacteria adhered to FUT1 transfected cells than to cells transfected with FUT3 or FUT4, to parental nontransfected cells, or to cells transfected with the plasmid vector only, as all of these controls lack cell surface expression of H(O) antigen (Fig. 4, B and C).


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Fig. 4.   CHO cells transfected with the human alpha 1,2-fucosyltransferase gene (FUT1) produce cell surface H-2 antigen, which was sufficient to induce campylobacter binding. Panel A, cell surface expression of H-2 antigen. Secondary anti-mouse, anti-H-2 monoclonal antibody, labeled with fluorescein (green), bound strongly to FUT1-transfected CHO cells counterstained with Evans blue (red), combining to appear orange. In contrast, anti-Lex, -Ley, -H-1, -Lea, or -Leb did not bind to FUT 1-transfected CHO cells. Panel B1, because parental CHO cells lack the human H-type gene and do not express fucosyl alpha 1,2-glycopeptides (including H-2) on the cell surface, few campylobacter bound to these cells. Panel B2, FUT1-transfected CHO cells are associated with copious numbers of invasive C. jejuni, strain 287ip. The bacteria in the center of the micrograph are associated with the cytoplasm of a cell that extends from the top of the micrograph to the bottom and to the left margin; because this cell is stretched, the cytoplasm is thinner than that of the other cells shown, and thus is a lighter shade of brown. Both light micrographs are of cells stained with Warthin-Starry. Panels C, scanning electron micrograph demonstrating binding of campylobacter to CHO cells transfected with FUT1. Panel C1, the control cells are transfected with only the plasmid vector and are not bound by campylobacter. Panel C2, the campylobacter are the corkscrew-like spiral bacteria associated within the surface of the transfected CHO cells.

The specificity of campylobacter binding to alpha 1,2 linkages of the FUT1-transfected CHO cells was further tested with agents that inhibit H-2 attachment (Table I). When the cells were pretreated with the lectins L. tetragonolobus or UEA I, or mAbs to H-2 antigen, agents that bind specifically to H-2, the binding by campylobacter was inhibited. In contrast, when they were pretreated with mAbs to H-1 and A antigens or the lectin D. biflorus (Table I), agents whose binding is independent of H-2, the campylobacter binding was not inhibited. This campylobacter binding to CHO-FUT1 cells was also inhibited by soluble mimetics of the H-2 ligand, such as neoglycoprotein containing the alpha 1,2-fucosylated ligand, human milk oligosaccharides, and, to a lesser extent, by 2'-fucosyllactose, all molecules that can bind to the campylobacter adhesin, making it unavailable to bind with cell surface H-2. The related molecules tri-Lex neoglycoprotein, lactose, and BSA, which do not bind specifically to H-2, did not inhibit (Table I). These data demonstrate the specificity for the binding between campylobacter and host cell H ligands and demonstrate that host cell expression of H-2 is sufficient to cause campylobacter adherence in most of the strains. This is compelling evidence that binding to the H epitopes is essential for attachment by invasive strains of C. jejuni to their host cells.


                              
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Table I
IC50 and IC95 values of alpha 1, 2-fucosyl ligands and homologs for the inhibition of campylobacter binding to CHO-FUT1 cells

Cell Agglutination Assays-- To confirm that the alpha 1,2-fucosyltransferase gene is the specific determinant for campylobacter binding, and to investigate the strain specificity of campylobacter binding to transfected CHO cells, the agglutination of several strains of campylobacter to various transfected CHO cells was investigated. Of the transfects for alpha 1,2-fucosyltransferase (CHO-FUT1), alpha 1,3- and alpha 1,4-fucosyltrasferase (CHO-FUT3), alpha 1,3-fucosyltransferase (CHO-FUT4), and parental CHO-V, only the CHO-FUT1 cells bound to campylobacter. The FUT1 transfect, whose expression of fucosyl alpha 1,2-linked determinants was confirmed as above (Table I) bound to all four of the pathogenic, adherent campylobacter strains tested (287ip, 84sp, 166ip, and 10sp), but did not bind to the nonpathogenic, non-adherent strains 50sp and 57sp. This confirms the specificity of pathogenic campylobacter binding to alpha 1,2-linked fucose (Table II).


                              
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Table II
Agglutination of parental CHO cells (CHO-V) and CHO cells
Cells were transfected with human fucosyltransferase genes alpha 1,2-fucosyltransferase (CHO-FUT1), alpha 1,3/4-fucosyltransferase (CHO-FUT3), and alpha 1,3-fucosyltransferase (CHO-FUT4) upon exposure to pathogenic and nonpathogenic campylobacter strains or UEA 1 lectin. Scores: 3+ = strong agglutination; 2+ = intermediate agglutination; 1+ = perceptible but weak agglutination.

Effect of Milk Oligosaccharides on Intestinal Colonization by Campylobacter-- The relevance of inhibition of H-2-mediated campylobacter binding was tested using the mixture of neutral oligosaccharides isolated from human milk, a natural source of H-2 and other H-Lewis epitopes. The ability of milk oligosaccharides to inhibit campylobacter infection of the gut was tested in three models of intestinal colonization: The first, a mouse model (32), was used to determine whether the human milk oligosaccharide fraction inhibits gut colonization in vivo. Mice given neutral human milk oligosaccharides by oral intubation had significantly less campylobacter colonization at both low (104 CFU/ml) and high (108 CFU/ml) inocula (p = 0.001 and 0.01, respectively) (Fig. 5A).


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Fig. 5.   Human milk oligosaccharides inhibit campylobacter colonization, both in mice and in human intestinal epithelium. Panel A, 2-week-old BALB/c mice were challenged with 104 (left) or 108 (right) CFU of C. jejuni invasive strain 287ip. Half of the mice were given milk oligosaccharides by oral intubation (filled circles), and half received PBS (open circles) (20). Each of the four treatment groups consisted of six mice. Those fed oligosaccharides displayed significant reductions of 4.1 and 2.6 logs of colonic campylobacter shedding in response to 104 and 108 bacterial inocula, respectively. Panel B, preincubation of campylobacter with milk oligosaccharides at one-half the concentration naturally found in human milk reduced colonization on fresh human intestinal (ileal) specimens by pathogenic strain 287ip (93% inhibition) and nonpathogenic 57sp (82% inhibition). In contrast, 2'-fucosyllactose inhibited strain 287ip by 70%, but did not inhibit the non-pathogenic 57sp. This is consistent with the pathogenic strain binding to 2-linked fucose and the nonpathogenic strain binding to some other epitope that is also present in human milk oligosaccharides.

In the second model, the ability of the human milk fucosyloligosaccharide fraction to inhibit campylobacter adherence in humans was determined ex vivo using fresh human intestinal mucosa. The neutral oligosaccharide fraction at one-half its natural concentration in pooled human milk inhibited colonization by the pathogenic campylobacter strain 287ip by 93%, and treatment with 2'-fucosyllactose alone caused 69% inhibition (p < 0.01; Fig. 5B). The nonpathogenic 57sp strain colonized human intestine at only half the rate as the pathogenic strain; treatment with neutral oligosaccharides reduced this binding by 82%, but little inhibition was observed following treatment of the nonpathogenic strain with 2'-fucosyllactose. These data are consistent with the pathogenic 287ip strain binding to a 2'-fucosyl moiety in the target cell, and the nonpathogenic 57sp binding with less avidity to a different moiety that is inhibited by components of the milk oligosaccharide mixture other than those containing the H-2 epitope.

A third model tested campylobacter colonization in pups of transgenic mice carrying the FUT1 human fucosyltransferase gene. These lactating mice expressed fucosyl alpha 1,2 ligand primarily in mammary gland tissue and, consequently, their milk contained H-2 blood group antigen (33). Each of their pups was challenged with inocula of 107, 106, or 102 CFU of pathogenic C. jejuni strain 287ip. Wild-type mice inoculated with 106 CFU of campylobacter generally remained infected throughout the 15 days of the study, with the percentage of infected animals ranging from 67 to 100% per time point. In contrast, the percentage of infected pups nursing transgenic dams decreased significantly over the course of the study (p = 0.0001). None of the pups inoculated with 102 CFU were infected after 5 days of nursing, and none of the pups challenged with 107 or 106 CFU campylobacter were infected after 9 days of nursing (Fig. 6). Intestinal clearance of campylobacter was significantly greater in pups nursing transgenic versus nontransgenic dams when the pups were challenged with identical inocula (106) (Mann-Whitney test, p = 0.006). Furthermore, each transgenic treatment group was significantly different from the nontransgenic group, regardless of inoculum (analysis of variance, p < 0.0001).


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Fig. 6.   Expression of alpha 1,2-fucosylglycoconjugates in milk of transgenic mice protects their nursing pups from intestinal colonization by campylobacter. BG/SJL mice were transfected with WAP promoter upstream from the human alpha 1,2-fucosyltransferase gene (FUT1), and their nursing pups were inoculated with 102, 106, and 107 CFU of pathogenic campylobacter per pup (i.e. 10 µl of 104, 108, or 109 CFU/ml). Control pups of nontransgenic dams were inoculated with 106 CFU. Every third day, one pup from each litter was sacrificed and tested for campylobacter intestinal colonization. Degree of colonization is expressed as the percentage of pups colonized in each group at each time post-inoculation. Shaded areas indicate the period during which an appreciable number of mice are infected. A statistically significant difference was observed when comparing the transgenic and the control (nontransgenic) groups (analysis of variance followed by examination of the underlying regression corresponding to the model; p < .0001).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Our findings indicate that epithelial cell surface H blood group (fucosyl alpha 1,2) epitopes serve as ligands tethering campylobacter to the intestinal mucosa in the essential first step of infection. Cell surface glycoconjugates have been implicated as mediators of cell-to-cell interactions, such as host cell adhesion, leukocyte adhesion and diapedesis. They also mediate adhesion by microbes (37), as pathogenic bacteria have adapted to utilize these cell surface structures for attachment and invasion (6). In this way, campylobacter appears to exploit H(O) antigen, which is located in the intestinal mucosa and widely distributed among humans, to attach to and invade host cells. The H-antigen is a fucosylated structure (Fucalpha 1,2Galbeta 1,3/4GlcNAc ... ) whose expression is determined by the presence of either of two different alpha 1,2-fucosyltransferases regulated primarily by the gene FUT1 (H) in red blood cells and vascular endothelium and by the gene FUT2 (Se) in exocrine secretions and intestinal mucosa (38, 39). Although H-antigen participates in adhesive events during embryonic development, tissue morphogenesis, cellular differentiation, and tumor metastasis (40), its role in cell function is not fully defined.

Human milk contains large amounts of fucosylated oligosaccharides with a wide array of structures, and the milk of most mothers contains alpha 1,2-linked fucosyloligosaccharides as a prominent feature. The expression of the alpha 1,2-linked epitope is determined by the presence of an active alpha 1,2-fucosyltransferase in the mammary epithelium, which occurs in individuals with the dominant form of the secretor (FUT2) gene. The results presented here add to growing evidence that such fucosylated structures may be major components of the innate immune system of human milk. In the present investigation, they appear to serve as soluble ligands that compete with intestinal epithelial cell surface receptors for binding to campylobacter and other pathogens that target alpha 1,2 ligands.

Our solid phase assay confirmed the avidity of campylobacter binding to blood group antigens. The most virulent campylobacter strains bound to several related fucose-containing blood group antigens, but bound most strongly to H ligands. The ability of H moieties to act as campylobacter ligands was then confirmed in CHO cells transfected with the human alpha 1,2-fucosyltransferase gene, FUT1, and expressing the fucosyl alpha 1,2 phenotype. Pathogenic campylobacter strains bound to these cells, but not to identical cells transfected with a transferase gene that does not catalyze production of fucosyl alpha 1,2-linkages or carrying the plasmid vector alone. Agents that specifically bind to H-2, such as mAbs, UEA I and Lotus tetragonolobus lectins, inhibited campylobacter binding to FUT1-transfected CHO cells, confirming that binding by pathogenic campylobacter requires the specific expression and availability of H-2 on its target. Finally, campylobacter binding to the transfected cells was inhibited by soluble H-2 mimetics, e.g. H-2 neoglycoprotein, fucosylated oligosaccharides of human milk, and 2'-fucosyllactose, which presumably compete with the cell surface H-2 ligands by binding to the bacterium, inhibiting its ability to adhere to its receptor on the surface of transfected CHO cells. This finding establishes the specificity of campylobacter binding to H-2 antigen of the host cell.

Experiments on the binding of pathogenic campylobacter strain 287ip to intact human intestine employed the nonpathogenic strain 57sp as a control. This strain, isolated from an asymptomatic child, did not bind to HEp-2 cells but, surprisingly, bound to intact human intestinal mucosa (though to a lesser extent than did 287ip). Also unexpected was the inhibition of 57sp binding by the milk oligosaccharide fraction. This strain may bind to a different epitope in the intestine, and its binding may be inhibited by components of the milk oligosaccharide mixture other than those containing H-2 epitope. This explanation is supported by 2'-fucosyllactose inhibiting the binding by the pathogenic strain, but not inhibiting the binding by the nonpathogenic strain. Thus, other epitopes of the intestinal epithelium may be important for the binding of other bacteria, including nonpathogenic strains. We are currently investigating several other pathogen/epitope pairs that are inhibited by other human milk glycoconjugates.

The differential expression of H-2 blood group antigen at different sites in the gastrointestinal tract could explain features of the pathology of campylobacter diarrhea, e.g. localization of infection. Furthermore, the genotypes of any mother/infant dyad may lead to differences in infant intestinal expression of H(O) antigen and maternal expression of H(O) antigen in milk, and thus may define differences in individual infant's risk of campylobacter infection. Thus, if expression of this antigen in intestinal mucosa is related to its expression in blood, an individual's Lewis blood group type might indicate his or her level of susceptibility to campylobacter (25). Heterogeneity of blood group glycan expression has been related to differential susceptibility to other pathogens. For example, epithelial cells from individuals of the recessive p blood group type do not express a receptor for uropathogenic Escherichia coli (41, 42), and these people are resistant to urinary tract infections. Individuals who express less of the P blood group glycolipids in their erythrocytes more frequently develop hemolytic uremic syndrome (43). Nonsecretors, i.e. individuals who do not secrete certain fucosylated blood group antigens, are more susceptible to recurrent urinary tract infections (44), and to Candida albicans infections. In human volunteers, persons with H(O) phenotype were significantly more likely to become infected with Norwalk virus (45). Susceptibility to cholera is strongly related to ABO status (46); although the molecular basis for this phenomenon has not yet been established, our preliminary investigations indicate that Vibrio cholerae also binds specifically to H(O) blood group antigen.

We propose that the interaction of campylobacter with enterocytes entails a progression of events. The initial step, binding to the host cell H-2 epitope, may be common to several enteropathogens. Polyvalency of H-2 epitopes on the host cell surface may amplify the binding specificity and avidity for bacteria. Subsequent steps could be unique to each pathogen. Docking of campylobacter to an H-2-containing transmembrane glycoprotein is likely to trigger host cell events enabling campylobacter invasion, analogous to host cell events seen in other types of bacterial binding to enterocytes (12, 47).

If H-2 binding by campylobacter is an essential first step in the pathogenesis, then soluble ligands that contain the H-2 epitope would be expected to inhibit colonization and infection by this pathogen in vivo. Colonization of mice by pathogenic campylobacter was indeed reduced by several orders of magnitude if the innoculum was accompanied by human milk oligosaccharides.

In a model that more closely resembles the nursing infant, mice were transfected with a WAP vector containing the FUT1 gene that is specifically expressed in the mammary gland during lactation. Large amounts of H-2 antigen are produced in the milks of homozygous dominant transfected mice; the milk of wild-type mice do not contain measurable H-2 antigen. Pups do not express this transfect because WAP expresses only in the actively lactating mammary gland. Pups nursing these transfected dams were strongly protected against intestinal colonization by campylobacter, while those nursing wild-type dams were not. This result not only reinforces that H-2 acts as the receptor for campylobacter, but shows also that soluble ligands containing H-2 epitopes in milk can protect young mammals from campylobacter infection. This experiment also strongly supports the concept that the large amount of H-2 epitopes found in human milk may represent an important component of the innate immune system of milk that can protect nursing infants against pathogens whose initial step of infection is contact with and adherence to intestinal mucosa.

Our first mouse model confirms that human milk oligosaccharides contain structures that inhibit binding of campylobacter to its target cell in vivo. The protective activity resides in fucosyl oligosaccharides alpha 1,2 structures, whose epitopes are shared by several blood group antigens. In human milk, the production of such fucosyl has long been attributed principally to the product of the secretor (FUT2) gene, another 2-fucosyltransferase. People with functional FUT2 have appreciable 2-linked fucosylated structures in their secretions, which include milk. However, homozygous recessive individuals for this trait do not produce appreciable fucosylated alpha 1,2 structures in their milk. We are currently studying whether the breastfeeding children of mothers who produce less alpha 1,2 oligosaccharides and glycoconjugates in their milk are at higher risk for campylobacter-associated diarrhea.

Our finding that the neutral, fucosylated oligosaccharide fraction of human milk inhibits campylobacter binding to its target cells adds to the body of knowledge concerning the disease-specific, protective, nonimmunoglobulin components of human milk. Previous studies showed that the nonimmunoglobulin fractions of human milk protect against various gastrointestinal, respiratory, and urinary tract infections in breastfed children, and that this protection may be attributed to the pathogen-blocking activity of milk oligosaccharides (48, 49). These oligosaccharides and glycoconjugates, which probably number in the hundreds, if not thousands, also inhibit secretory diarrhea caused by the stable toxin of E. coli (27, 50, 51), suggesting that human milk glycoconjugates include inhibitors of enteropathogens whose pathogenic mechanisms differ from those of campylobacter.

In conclusion, a critical human intestinal ligand for campylobacter contains H-2 antigen, and human milk contains fucosylated oligosaccharides that inhibit campylobacter binding. An H-2 carbohydrate moiety may be the essential component of human milk that acts as a soluble receptor for campylobacter, thereby inhibiting binding of campylobacter to its intestinal receptors, the essential first step of its pathogenesis. The fucosyloligosaccharide concentrations in human milk were ample to inhibit campylobacter binding in vitro, in human intestine ex vivo, and in mice in vivo. Futhermore, when H-2 glycoconjugates are expressed in the milk of transgenic mice, their nursing pups are strongly protected from infection by virulently pathogenic campylobacter. These results strongly suggest that oligosaccharides in human milk contribute to protection of infants against C. jejuni and other enteric pathogens. Synthetic mimics of these oligosaccharides may become a novel class of prophylactic and therapeutic antimicrobial agents with mechanisms distinct from the current generation of antibiotics.

    ACKNOWLEDGEMENTS

We thank Drs. Ardythe L. Morrow, Pedro A. Prieto, and Beatriz R. Ruiz-Palacios, and Ms. Kathryn Newburg for important contributions to this study.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant HD13021, by National Council for Science and Technology (Conacyt) (Mexico) Grant 1428, and a fellowship from Conacyt (to L. E. C.).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. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: Dept. of Infectious Diseases, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Vasco de Quiroga 15, Mexico D.F. 14000, Mexico. Tel.: 525-55655-9675; Fax: 525-55513-0010; E-mail: gmrps@servidor.unam.mx.

Published, JBC Papers in Press, January 31, 2003, DOI 10.1074/jbc.M207744200

    ABBREVIATIONS

The abbreviations used are: UEA I, Ulex europaeus agglutinin I; PBS, phosphate-buffered saline; CFU, colony-forming units; TBS, Tris-buffered saline; DIG, digoxigenin; CHO, Chinese hamster ovary; mAbs, monoclonal antibodies; BSA, bovine serum albumin; FUT1, alpha 1,2-fucosyltransferase gene.

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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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