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INTRODUCTION |
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
1,2-FUT1 inhibits
campylobacter colonization in the intestine of their nursing pups. pWAP
1,2-FUT1 expresses H (O) antigen mainly in mammary gland
during lactation.
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EXPERIMENTAL PROCEDURES |
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
-naphthol/H2O2/TBS (15 ml of
TBS, 3 ml of MeOH, 9 mg of
-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
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
1,2-fucosyltransferase (CHO-FUT1),
1,3/4-fucoslytransferase (CHO-FUT3), and
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
1,2-fucosyltransferase needed for the synthesis of the
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--
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
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
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.
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RESULTS |
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.
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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
1,2-linked fucose, with especially high avidity for the
Fuc
1,2Gal
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.
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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.
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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
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
1,2 moiety is a critical feature for
campylobacter binding, and that the H-2 epitope is the preferred
fucosyl
1,2 structure.
Specificity of H-2 Binding by Campylobacter in Transfected
Cells--
To test the hypothesis that cellular expression of
1,2-fucosyl moieties (H antigen) allows campylobacter binding, CHO
cells were transfected with the FUT1 gene for human
1,2-fucosyltransferase whose expression product catalyzes the final
step in the synthesis of H antigen. Parental CHO cells do not express
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
1,2-fucosyltransferase human gene
confers on CHO cells the ability to complete the synthesis of all
fucosyl
1,2-containing antigens; the actual products will depend
upon the availability of endogenous substrates. Identical cells were
likewise transfected with
1,3/4-fucosyltransferase (the Lewis gene,
FUT3), with human
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
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 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.
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The specificity of campylobacter binding to
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
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 1, 2-fucosyl ligands and
homologs for the inhibition of campylobacter binding to CHO-FUT1 cells
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Cell Agglutination Assays--
To confirm that the
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
1,2-fucosyltransferase
(CHO-FUT1),
1,3- and
1,4-fucosyltrasferase (CHO-FUT3),
1,3-fucosyltransferase (CHO-FUT4),
and parental CHO-V, only the CHO-FUT1 cells bound to
campylobacter. The FUT1 transfect, whose expression of
fucosyl
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
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
1,2-fucosyltransferase (CHO-FUT1), 1,3/4-fucosyltransferase
(CHO-FUT3), and 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.
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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.
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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
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
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 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).
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DISCUSSION |
Our findings indicate that epithelial cell surface H blood group
(fucosyl
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
(Fuc
1,2Gal
1,3/4GlcNAc ... ) whose expression is determined by the presence of either of two different
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
1,2-linked fucosyloligosaccharides as a prominent feature. The
expression of the
1,2-linked epitope is determined by the presence
of an active
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
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
1,2-fucosyltransferase gene, FUT1, and
expressing the fucosyl
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
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
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
1,2 structures in their milk. We are currently studying
whether the breastfeeding children of mothers who produce less
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.