Probiotics inhibit enteropathogenic E. coli
adherence in vitro by inducing intestinal mucin gene
expression
David R.
Mack1,2,
Sonia
Michail1,2,
Shu
Wei1,2,
Laura
McDougall1,2, and
Michael A.
Hollingsworth3
1 Combined Section of Pediatric
Gastroenterology and Nutrition,
Department of Pediatrics
2 Center for Human Nutrition, and
3 Eppley Science and Allied
Health, University of Nebraska Medical Center, Omaha, Nebraska 68198
 |
ABSTRACT |
Probiotic agents,
live microorganisms with beneficial effects for the host, may offer an
alternative to conventional antimicrobials in the treatment and
prevention of enteric infections. The probiotic agents
Lactobacillus plantarum 299v and
Lactobacillus rhamnosus GG
quantitatively inhibited the adherence of an attaching and effacing
pathogenic Escherichia coli to HT-29
intestinal epithelial cells but did not inhibit adherence to
nonintestinal HEp-2 cells. HT-29 cells were grown under conditions that
induced high levels of either MUC2 or MUC3 mRNA, but HEp-2 cells
expressed only minimal levels of MUC2 and no MUC3 mRNA. Media enriched
for MUC2 and MUC3 mucin were added exogenously to binding assays and
were shown to be capable of inhibiting enteropathogen adherence to
HEp-2 cells. Incubation of L. plantarum 299v with HT-29 cells increased MUC2 and MUC3
mRNA expression levels. From these in vitro studies, we propose the
hypothesis that the ability of probiotic agents to inhibit adherence of
attaching and effacing organisms to intestinal epithelial cells is
mediated through their ability to increase expression of MUC2 and MUC3
intestinal mucins.
MUC2; MUC3; Escherichia coli
O157:H7; Lactobacillus plantarum; Lactobacillus rhamnosus
 |
INTRODUCTION |
THE EPITHELIAL CELL layer of the intestinal tract is
strategically located between the many microbes and antigens of the
intestinal lumen and the inflammatory and immune effector cells of the
host's lamina propria. The intestinal epithelial cell is capable of a regulated marked production of selected chemokines in response to
invasive bacteria (39) and, as such, may be an important component in
development of the host innate and acquired immune responses.
Noninvasive enteropathogens, such as enterohemorrhagic Escherichia coli (EHEC) serotype
O157:H7, also elicit increases in cytokines, such as interleukin-8,
although not to the same magnitude as invasive pathogens (16). Another
noninvasive enteric pathogen, enteropathogenic E. coli (EPEC), has been demonstrated to stimulate
transepithelial migration of neutrophils (33) and cytokines such as
interleukin-8 through the activation of the nuclear transcription
factor (NF)-
B of infected cells (34). Furthermore, EPEC can produce
factors that regulate the function of the intestinal mucosal immune
system (19). Taken together, these findings suggest that there may be
other protective responses provided by epithelial cells.
EHEC serotype O157:H7 and EPEC belong to a group of enteric pathogens
that contain a pathogenicity island on the bacterial chromosome that
induces cytoskeletal rearrangements in infected epithelial cells
leading to the formation of a characteristic attachment and effacement
lesion (28). Intestinal epithelial cell-derived mucins from animals
bind to human EHEC and inhibit animal and human EPEC strain adherence
in vitro (7). The binding of pathogens by mucosal epithelial cell
mucins is an important defense mechanism for the host (7).
Mucins are high-molecular-weight glycoproteins synthesized and secreted
by epithelial cells of a number of organ systems, including the
intestinal tract. Mucins are characterized by their large size, high
content of carbohydrates, and
O-glycosidic bonds between
N-acetylgalactosamine and either
serine or threonine in the peptide backbone (7). Different mucin genes
have been identified by isolation of partial cDNAs containing unique
tandem repeat domains. Among the different human mucin genes, MUC2 and
MUC3 are the predominant ileocolonic mucins. The MUC2 mucin polypeptide contains a repetitive peptide of 23 amino acids that is rich in threonine and proline residues, is heavily glycosylated, and is flanked
by cysteine-rich domains (10). The MUC2 gene is expressed in goblet
cells of the small and large intestine (3), and MUC2 mucins may be the
major secreted mucin component of the colon (12, 38). In contrast, MUC3
has a 17-amino acid tandem repeat rich in threonine and serine residues
with a cysteine-rich carboxy-terminal domain that shows homology to
epidermal growth factor (11). MUC3 is not highly expressed in the colon
(12, 38) but shows expression in both goblet cells and enterocytes of
the small intestine (3).
Enteric pathogens possess a number of strategies to circumvent mucins
that overlay epithelial cells (7). Mucin-pathogen interactions may be
determined by the quantity and quality of epithelial cell mucins. Live,
nonpathogenic bacteria that are fed to humans (i.e., probiotics) have
been shown to prevent and/or improve intestinal infections with
pathogens (5). Hypothesized mechanisms of colonization resistance by
probiotic agents include direct actions against pathogens or their
receptors and stimulation of the epithelial cell host acquired immune
response (5). In this study, we sought to investigate interactions
between probiotics and MUC2 and MUC3 intestinal mucins. The role of
probiotic agents in inhibition of binding of attaching and effacing
enteric pathogens with intestinal epithelial cells was investigated in
an in vitro model.
 |
MATERIALS AND METHODS |
Bacteria and growth conditions. Stock
cultures of EPEC strain E2348/69 (serotype O127:H6) and EHEC strain CL8
(serotype O157:H7) were maintained on trypticase soy agar slants
(Becton-Dickenson Microbiology Systems, Cockeysville, MD) at 4°C.
Stock cultures of Lactobacillus
plantarum strain 299v and
Lactobacillus rhamnosus strain GG
(American Type Culture Collection 53103, Rockville, MD) were maintained
on MRS agar (Difco Laboratories, Detroit, MI) at 4°C.
Bacteria were kindly provided by Dr. James Kaper (E2348/69), Center for
Vaccine Development (Baltimore, MD), Dr. Philip Sherman (CL8), The
Hospital for Sick Children (Toronto, Canada), and Drs. Stig Bengmark
and Bengt Jeppson (299v), Lund University (Lund, Sweden).
EPEC strain E2348/69 was originally isolated during an outbreak of
infant diarrhea (20), and EHEC strain CL8 was isolated from stools of a
child with hemorrhagic colitis and hemolytic uremic syndrome (4). These
strains cause the attachment/effacement lesion and contain the
eaeA gene (4, 14). EPEC and EHEC
strains that cause the attachment/effacement lesion have a conserved
genetic locus of enterocyte effacement (28).
L. rhamnosus strain GG was isolated in
vitro from stool specimens of healthy humans (8). L. plantarum strain 299v came from sourdough and has
phenotypic and genotypic similarity to a strain isolated in vivo from
the human intestinal tract (15). Both
Lactobacillus strains show the ability
to colonize the human intestinal tract after oral administration (15,
35).
E. coli strains were grown overnight
at 37°C in static, nonaerated Penassay broth (Difco), and
Lactobacilli strains were grown overnight at 37°C in static, nonaerated MRS broth (Difco) to reach the mid-log growth phase. Bacteria were harvested by centrifugation at
2,500 g for 15 min at 20°C in a
GPR centrifuge (Beckman Instruments, Palo Alto, CA). After two washes
in sterile pH 7.4 Dulbecco's PBS (Life Technologies, Gaithersburg, MD)
at 25°C, bacteria were resuspended in PBS. Quantification of
bacterial suspensions was determined by using a standard curve (data
not shown) of bacterial colony-forming units (CFU) on MacConkey agar
(Difco) or MRS agar relative to visible absorbance (600 nm; Spectronic
Genesys 5 spectrophotometer, Rochester, NY).
Cell growth conditions. HT-29 colonic
adenocarcinoma cells (American Type Culture Collection) were grown in
McCoy's 5a culture medium (Life Technologies). Some HT-29 cells were
cultured under separate growth conditions to increase MUC3 mRNA
expression and reduce MUC2 mRNA expression relative to HT-29 cells
grown in original culture conditions (25). This was accomplished
through a progressive transfer of some HT-29 cells from the regular
McCoy's 5a culture medium to a glucose-free, 5 mM galactose-containing
McCoy's 5a culture medium as previously described (25). HEp-2 human
laryngeal epidermoid carcinoma cells (American Type Culture Collection) were grown in MEM (Life Technologies). Culture media for the various cell lines were supplemented with 10% heat-inactivated qualified fetal
bovine serum (Life Technologies) and antibiotics (100 U/ml penicillin
G, 100 mg/ml streptomycin sulfate, and 0.25 mg/ml amphotericin B; Life
Technologies). The cultures were grown at 37°C in a humidified atmosphere with 5% CO2 and were
passaged after washing with Earle's balanced salt solution (Life
Technologies) using either trypsin (HT-29 cells) or trypsin-EDTA (HEp-2
cells; Life Technologies).
Probiotic inhibition assays.
Alteration in the adherence to epithelial cells of EPEC strain E2348/69
and EHEC strain CL8 by L. plantarum
strain 299v was determined using modifications of an in vitro
epithelial binding assay described previously (37).
For the in vitro adherence inhibition assay, 6 × 105 epithelial cells (HEp-2 or
HT-29) were suspended in the appropriate antibiotic-free culture medium
and transferred to individual wells of a 12-well polystyrene tray
(Fisher). Cells were grown to confluence and washed three times with
sterile, 37°C Hanks' balanced salt solution (Life Technologies) to
remove culture medium and nonattached cells. Bacteria were added to 2.1 ml of the appropriate antibiotic-free cell growth medium. Different
numbers of L. plantarum 299v in a
0.1-ml volume of PBS (pH 7.4, 25°C) were added 1 h before
(preincubation studies) or at the same time (coincubation studies) as
105 CFU (in 0.1 ml of PBS, pH 7.4, 25°C) of a pathogenic E. coli strain of bacteria. After incubation for 3 h at 37°C,
cells were washed four times with Dulbecco's PBS (pH 7.4, 37°C) to
remove nonadherent bacteria. Cells with adherent bacteria were released from polystyrene wells using 0.1 ml of trypsin-EDTA (HEp-2 cells) or
trypsin alone (HT-29 cells). After 10 min incubation at 37°C, ice-cold sterile PBS was added to each well, and the well contents were
agitated to dissociate epithelial cells. Serial dilutions of adherent
bacteria were plated on MacConkey agar and incubated overnight at
37°C for subsequent quantification by counting CFU. All experiments
were run in triplicate.
Supernatants from the growth of
Lactobacilli that were collected were
filtered through a 0.2-µm filter (Gelman Sciences, Ann Arbor, MI) and
then boiled for 10 min. Plating on MRS agar plates showed no evidence
of bacterial growth following these procedures. In some experiments,
these cell-free sterile supernatants from overnight growth of
L. plantarum 299v were added in place
of the L. plantarum 299v. Various
dilutions of the supernatant in a total volume of 1 ml were added to
HT-29 cells 1 h before the addition of
106/0.1 ml E. coli strain E2348/69. EPEC E2348/69 were added to 1.1 ml of the cell growth medium overlying the cells and were incubated for
3 h. Controls had 1 ml of MRS broth added.
In preliminary experiments, we evaluated the viability of
E. coli coincubated with
L. plantarum 299v. After harvesting of bacteria, EPEC strain E2348/69 were mixed with antibiotic-free McCoy's
5a culture medium and were added to 12-well plates without epithelial
cells to reach a final concentration of
105/well. To one-half of the
wells, 109/well
L. plantarum 299v were added. After a
3-h incubation, quantification was made by CFU determinations after
serial dilutions. No difference (P = 0.37) was observed
between the number of E. coli CFU
between those wells that were or were not coincubated with
L. plantarum 299v. A crystal violet
dye-binding assay (2) was also used in preliminary experiments to
estimate the number of HT-29 cells after bacterial incubation.
Identical cell numbers were quantified after a 3-h incubation of 1 × 105 E2348/69/well (8.9 × 105 ± 2.9 × 104 cells/well) compared with
coincubating 1 × 105 EPEC
and 1 × 109
L. plantarum 299v (1.0 × 106 ± 4.4 × 104 cells/well,
P = 0.1) for 3 h. Culture media
overlying HT-29 cells and HEp-2 cells contained phenol red to monitor
pH. At the end of the incubation period with
109 L. plantarum 299v, the pH was turning acidic. The pH in
the media overlying both HT-29 cells and HEp-2 cells were not different from each other.
Isolation of mucins. Cell culture
supernatants overlying HT-29 cells grown in glucose culture medium to
express high levels of MUC2 mRNA or HT-29 cells grown in galactose
culture medium to express high levels of MUC3 mRNA (25) were processed
by techniques previously described for purification of intestinal
goblet cell mucin (24, 26). To minimize proteolytic degradation of the constituents in the culture media, 5 mM
N-ethylmaleimide (Sigma-Aldrich), 2 mM
phenylmethylsulfonyl fluoride (Sigma-Aldrich), and 0.01% sodium azide
(Sigma-Aldrich) were added along with 5 mM EDTA to the collected cell
culture media. The culture media were centrifuged at 30,000 g for 30 min at 4°C to remove
pelleted cellular and particulate debris. Components of the soluble
supernatant were subdivided by buoyant density using isopycnic
ultracentrifugation in cesium chloride (Fisher) with a starting density
of 1.46 g/ml. The suspension was placed in polyallomer centrifuge tubes
(DuPont, Wilmington, DE) and centrifuged in a Sorvall 50.2 Ti rotor
(DuPont) at 105,000 g for 48 h at
4°C. After centrifugation, a needle was inserted to the bottom of
the centrifuge tube, and eight fractions of equal volume were collected
by using a peristaltic pump (Spectra/Chrom Macroflow pump; Spectrum
Medical Industries, Los Angeles, CA). The eight fractions were analyzed
with a refractometer (Abbe L; Milton Roy, Rochester, NY) to determine
the buoyant density. Fractions were then placed in wetted cellulose
dialysis tubing (50 kDa exclusion; Spectrum) and dialyzed against
deionized water for 48 h at 4°C. Each fraction then underwent
determination of nucleic acid content [optical density (OD) 260 nm], total protein by the method of Lowry with a
prepared concentration of albumin (fraction V; Sigma-Aldrich) used as
standard, and glycoprotein concentration using the periodic acid-Schiff
assay with a prepared concentration of crude porcine mucin
(Sigma-Aldrich) as the reference standard as previously described (24).
Samples were partially lyophilized and were stored at
70°C
until use in assays.
Mucin inhibition of bacterial adhesion
assay. Examination of the inhibitory effects of
cellular products secreted in their culture medium on binding of
E. coli strain E2348/69 to HEp-2 cells
was determined using modifications of the in vitro assay described
(37). Briefly, the HEp-2 cells grown to confluence with antibiotic-free
MEM in 12-well polystyrene trays (Fisher) were rinsed three times with
sterile, 37°C Earle's balanced salt solution (Life Technologies)
to remove culture medium and nonattached cells. Approximately 2.5 × 106 CFU
E. coli E2348/69 in 0.1 ml of PBS (pH
7.4), 100 µg protein in a total volume of 0.1 ml of the different
buoyant density fractions (dialyzed), and 2.1 ml antibiotic-free MEM
were added to each well. After incubation for 3 h at 37°C,
nonadherent bacteria were removed by six washes with PBS (pH 7.4, 37°C). Serial dilutions of adherent bacteria that had been
dissociated from HEp-2 cells were plated for quantification by counting
CFU. All experiments were run in triplicate.
Alteration of MUC2 and MUC3 mRNA expression by
bacteria. HT-29 cells were grown to confluence (Falcon
3028; Becton-Dickinson, Franklin Lakes, NJ) in either the
glucose-containing McCoy's 5a culture medium or glucose-free,
galactose-containing culture medium (25). To ensure that the same
relative number of bacteria was used in RNA harvest experiments as was
used in binding studies, the number of bacteria added to flasks was
based on cell culture site-contact surface area. That is, the surface
contact area of flasks was calculated to be 45 times greater than the
surface area of a well of 12-well plates. For studies using flasks, 45 times the number of bacteria were added than were added to each well of
the 12-well plate to ensure there was not a greater concentration of
bacteria per cell for the two systems. Therefore, 4.5 × 106 E. coli CFU/flask or 4.5 × 1010 L. plantarum 299v CFU/flask were added to culture flasks
and incubated for 1 h before collection of total cellular RNA. In some
experiments, 15 ml of a 1:25 dilution of sterile bacterial supernatant
from L. plantarum 299v mixed with 15 ml
of cell growth medium were added in place of the bacteria and were
incubated for 1 h before HT-29 cellular RNA collection.
Total RNA preparation. Total cellular
RNA was isolated from cells using a guanidine isothiocyanate-cesium
chloride cushion ultracentrifugation technique (25). Briefly, cells
were washed with Earle's balanced salt solution and were lysed with a
solution containing 4 M guanidine isothiocyanate (Fisher Scientific,
Fair Lawn, NJ), 50 mM sodium acetate (Sigma-Aldrich Chemical, St.
Louis, MO), and 250 mM 2-mercaptoethanol (Fisher). Total RNA was
recovered by centrifugation through 5.7 M cesium chloride (Amresco,
Solon, OH) and 0.025 M sodium acetate cushion in a SW 41 Ti rotor
(Beckman Instruments) at 32,000 rpm for 18 h at 20°C. The RNA
pellets were suspended in 0.3 M sodium acetate and were precipitated
with 2.5 vol of ice-cold ethanol.
Mucin and glyceraldehyde-3-phosphate dehydrogenase
probes. The MUC2 probe (clone SMUC41) and the MUC3
probe (clone SIB 124; kindly provided by Drs. James Gum and Young Kim)
cDNA inserts were cut from pBS-SK vectors with
EcoR I (Life Technologies), gel
purified, and labeled with
[
-32P]dCTP (3,000 Ci/mmol; Amersham Life Sciences, Arlington Heights, IL) using a random
primer labeling kit (Amersham). Unincorporated nucleotides were removed
by Sephadex-G50 (Pharmacia Biotech, Piscataway, NJ) filtration.
Selected Northern blots were also hybridized with a
glyceraldehyde-3-phosphate dehydrogenase probe to ensure there was no
degradation of mRNA samples. Specific activity was determined by
scintillation counting. Probes contained a minimum of 5 × 108
counts · min
1 · µg
DNA
1.
Northern blot analysis. Twenty
micrograms of total RNA of samples were subjected to electrophoresis on
1.2% agarose gels containing 0.66 M formaldehyde and ethidium bromide
to allow for ultraviolet light visualization of 28S and 18S RNA.
Transfer to nitrocellulose membrane was via capillary blotting. Probe
hybridization of Northern blots was carried out at 42°C for 18 h as
previously described (25). Signals corresponding to mucin and
glyceraldehyde-3-phosphate dehydrogenase expression were detected by
Phosphor screen autoradiography (Molecular Dynamics, Sunnyvale, CA).
Screens were scanned by a PhosphorImager (Molecular Dynamics) and were
quantified by area integration using ImageQuant software (version 3.3;
Molecular Dynamics). Relative levels of 28S RNA were measured using a
Computing Densitometer with ImageQuant software (version 3.3; Molecular Dynamics) from photographic negatives (type 665 film; Polaroid, Cambridge, MA) of agarose/formaldehyde gels taken under ultraviolet light.
Statistical analysis. Group data are
expressed as means ± SE. Analyses between two groups were
made by unpaired two-tailed t-tests,
and analyses between multiple groups were determined using one-factor
ANOVA with 95% confidence intervals. Post hoc ANOVA analyses were
determined by Fisher's protected least-significant difference using
the StatView version 4.0 software program (Abacus Concepts, Berkeley, CA).
 |
RESULTS |
Inhibition of adherence of E. coli by L. plantarum. Binding
of 105 CFU/well
E. coli E2348/69 to HT-29 cells was
evaluated in the presence of increasing numbers of L. plantarum 299v. As shown in Fig.
1, progressive inhibition of
E. coli E2348/69 was noted when
greater concentrations of L. plantarum
299v were added simultaneously to each well. In experiments in which
the L. plantarum 299v was preincubated
with the HT-29 cells for 1 h before the addition of E2348/69, a smaller
inoculum of L. plantarum 299v was
required to effect a significant reduction of E2348/69 epithelial cell adherence. That is, even 107
L. plantarum 299v/well (8.5 × 104 ± 9.4 × 103 E2348/69 CFU/well) inhibited
E2348/69 binding more than controls with no L. plantarum 299v/well (1.2 × 105 ± 1.5 × 104 E2348/69 CFU/well,
P < 0.05). However, when
EPEC E2348/69 was added to HT-29 cells and incubated for 3 h before the
addition of L. plantarum 299v for an
additional 2-h incubation, no reduction in EPEC binding was
demonstrated (data not shown).

View larger version (51K):
[in this window]
[in a new window]
|
Fig. 1.
Quantitative inhibition of enteropathogenic
Escherichia coli (EPEC) adherence to
HT-29 cells by Lactobacillus plantarum
299v. To each well of 12-well plates growing HT-29 cells, a 1 × 105 colony-forming unit (CFU)/well
inoculum of EPEC E2348/69 was added with increasing amounts of
L. plantarum 299v. After a 3-h
incubation, EPEC adherent to HT-29 cells were quantified by CFU
determinations as a percentage of adherent EPEC in wells on each plate
that were without added L. plantarum
299v. Results are shown as means ± SE of 8 triplicate
results (105 and
108,
n = 4 experiments). * Compared
with controls without the coincubated bacteria, there was decreased
binding of EPEC with 108 and
109 L. plantarum 299v/well (P < 0.05).
|
|
Similar to our findings with the EPEC strain we used, adherence of EHEC
strain CL8 to HT-29 cells was also decreased in the presence of
increasing numbers of L. plantarum
299v. Binding of EHEC strain CL8 to HT-29 cells with
108 L. plantarum 299v/well (1.0 × 104 ± 1.2 × 103 CL8 CFU/well) and
109 L. plantarum 299v/well (2.9 × 102 ± 3.3 × 101 CL8 CFU/well) was less than
with 107 L. plantarum 299v/well (1.6 × 104 ± 1.9 × 103 CL8 CFU/well),
105 L. plantarum 299v/well (1.6 × 104 ± 3.8 × 103 CL8 CFU/well), or no
L. plantarum/well (1.6 × 104 ± 1.9 × 103 CL8 CFU/well,
P < 0.05).
A 1-h preincubation with L. plantarum
before the addition of 105 CL8
also decreased the required inocula of L. plantarum 299v to inhibit binding of EHEC strain CL8 to
intestinal epithelial cells. Both
107 and
109 L. plantarum 299v/well inhibited binding compared with
controls without L. plantarum 299v
(P < 0.05) as shown in Fig.
2.

View larger version (39K):
[in this window]
[in a new window]
|
Fig. 2.
Quantitative inhibition of enterohemorrhagic E. coli (EHEC) adherence to HT-29 cells by
L. plantarum 299v. To each well of
12-well plates that contained HT-29 cells, a 1 × 105 CFU/well inoculum of EHEC
O157:H7 was added 1 h after the addition of increasing amounts of
L. plantarum 299v. Four hours after
EHEC were added, EHEC adherent to HT-29 cells were quantified by
determining CFU. Results are expressed as means ± SE of 12 triplicate results. * There was decreased binding of EHEC upon
coincubation of 107 and
109 L. plantarum 299v/well (P < 0.05).
|
|
In those experiments in which the target epithelial cell for EPEC
binding was HEp-2 cells, there was no quantitative inhibition with
increasing amounts of L. plantarum
299v. As shown in Fig. 3, the yields of
adhering EPEC after coincubation of
105 E2348/69/well with no
L. plantarum 299v and
105,
107, and
109 L. plantarum 299v/well were similar
(P > 0.05).

View larger version (48K):
[in this window]
[in a new window]
|
Fig. 3.
Adherence of EPEC to nonintestinal epithelial cells during coincubation
with L. plantarum 299v. To each well
of HEp-2 cells, 1 × 105
CFU/well of EPEC E2348/69 was added with increasing amounts of
L. plantarum 299v. After a 3-h
incubation, EPEC adherent to HEp-2 cells were quantified by determining
CFU. Results are expressed as means ± SE of 15 triplicate results.
There were no differences in EPEC binding to these nonintestinal
epithelial cells on coincubation with the indicated amounts of
L. plantarum 299v (P > 0.05).
|
|
Dilutions of sterile supernatants collected after overnight growth of
L. plantarum 299v were added to HT-29
cell culture media for 1 h followed by the addition of a
106 E2348/69 inoculum/well for a
3-h incubation. Adherent EPEC were then quantified as previously
described. As shown in Fig. 4, undiluted supernatants and supernatants that were diluted 10-fold inhibited EPEC
binding compared with supernatants diluted 100-fold and control wells
without supernatant added (P < 0.05). Similar results were obtained using HT-29 cells grown in
galactose-containing culture medium to increase MUC3 expression.

View larger version (42K):
[in this window]
[in a new window]
|
Fig. 4.
Quantitative inhibition of EPEC adherence to HT-29 cells by sterile
L. plantarum 299v supernatants. To
each well of HT-29 cells that various dilutions of spent MRS broth of
L. plantarum 299v culture had been
added for 1 h was added 1 × 106 CFU/well of EPEC strain
E2348/69. After a 3-h incubation of EPEC, bacteria adherent to HT-29
cells were quantified by CFU determinations. Results are means ± SE
of 6 triplicate experiments. * There was decreased HT-29 cell
EPEC binding for 1:10 dilution and undiluted supernatants compared with
1:100 dilution and controls without supernatant added
(P < 0.05).
|
|
To determine whether L. plantarum 299v
was unique in inhibiting adhesion of E2348/69 to HT-29 cells, another
human probiotic agent, L. rhamnosus
strain GG, was also used in some E2348/69 coincubation experiments.
Both 109 L. plantarum 299v/well and
109 L. rhamnosus strain GG/well inhibited E2348/69 binding
compared with control EPEC E2348/69 binding without
Lactobacillus coincubation (P < 0.05) but were not different
from each other in inhibiting EPEC binding to HT-29 cells
(P > 0.05), as shown in Fig.
5.

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 5.
Inhibition of EPEC adherence to HT-29 cells by probiotic agents. Each
well of HT-29 cells received 1 × 105 CFU/well of EPEC E2348/69;
some wells also received 1 × 109 of either L. plantarum 299v (Lp299v) or
Lactobacillus rhamnosus GG (LrGG).
After a 3-h incubation, EPEC adherent to cells were quantified by
determining CFU. Results are expressed as means ± SE of 12 independent experiments. * Both Lp299v and LrGG decreased binding
of EPEC compared with controls (P < 0.05); however, those reductions were similar to each other
(P > 0.05).
|
|
MUC2 and MUC3 mRNA expression.
Northern blots hybridized with MUC2 and MUC3 cDNA probes and 28S RNA
levels of the blots are shown in Fig. 6.
HT-29 cells grown in the glucose-containing culture medium showed high
MUC2 and low MUC3 mRNA expression. The opposite pattern was seen in
HT-29 cells grown in the glucose-free, galactose-containing culture
medium in which a high MUC3 and low MUC2 mRNA expression was found. The
MUC2 mRNA and MUC3 mRNA pattern detected by autoradiography is similar
to that seen previously (25). HEp-2 cells expressed minimal levels of
MUC2 mRNA, but MUC3 mRNA was not detected (Fig. 6). In separate
experiments, there was no increased MUC2 mRNA or MUC3 mRNA expression
detected in HEp-2 cells incubated with the equivalent number of
109/well of L. plantarum 299v (data not shown).

View larger version (88K):
[in this window]
[in a new window]
|
Fig. 6.
Northern blot and analysis of MUC2 and MUC3 mRNA expression levels.
Total RNA (20 µg/lane) from HT-29 cells grown in glucose-containing
media (lanes 1 and
4), HT-29 cells grown in
galactose-containing media (lanes 2 and 5), and HEp-2 cells
(lanes 3 and
6) were separated by electrophoresis
on 1.2% agarose/formaldehyde gels containing ethidium bromide. RNA was
transferred to nitrocellulose membranes, and 28S RNA was visualized
under ultraviolet light (A and
C). Hybridization with a
random-primed
[32P]cDNA probe of the
MUC2 tandem repeat (B) or the MUC3
tandem repeat (D) was detected by
autoradiography for 13 h at 70°C. HT-29 cells grown in
glucose-containing media had high MUC2 mRNA expression, and HT-29 cells
grown in glucose-free galactose-containing media had high MUC3 mRNA
expression. HEp-2 cells showed minimal MUC2 mRNA expression and no MUC3
mRNA expression.
|
|
Inhibition assays with mucins. To
examine whether MUC2 and MUC3 mucins added to the bacterial adhesion
assay could inhibit EPEC adherence to HEp-2 cells, pooled supernatants
from HT-29 cells were collected. Densities of the eight separated
fractions of pooled supernatants from MUC2-enriched HT-29 cells grown
in glucose culture medium ranged from 1.337 to 1.510 g/ml and from 1.338 to 1.506 g/ml for supernatant of MUC3-enriched HT-29 cells grown
in glucose-free, galactose-containing culture medium. As previously for
mucins from intestinal mucosa (24, 26), the most dense cesium chloride
fraction (fraction 1) had a
relatively high nucleic acid content (OD 260 nm > 0.45) compared with
the middle-density fractions (OD 260 nm < 0.3). Comparable protein and glycoprotein profiles were obtained with corresponding buoyant density fractions, with progressively greater protein amounts found in
lighter buoyant density fractions. Glycoprotein content peaked in the
middle fractions and was similar to previous characterizations of
mucosal epithelial cell-derived mucins (24, 26, 37).
Quantitative inhibition of EPEC binding to HEp-2 cells was observed
with material derived from buoyant density fraction
4 of MUC3-enriched mucin. This fraction had a density
and a glycoprotein-to-protein ratio consistent with mucin glycoproteins
(24, 26). As shown in Fig. 7, quantitative
inhibition of EPEC E2348/69 (2.5 × 106 bacteria/well) was
demonstrated (P < 0.05).

View larger version (34K):
[in this window]
[in a new window]
|
Fig. 7.
Quantitative inhibition of EPEC adherence by mucin. To each well of
HEp-2 cells, 2.5 × 106
CFU/well of EPEC E2348/69 were added with increasing amounts of
material (protein) derived from MUC3-enriched buoyant density
fraction 4. After a 3-h incubation,
EPEC adherent to HEp-2 cells were quantified by determining CFU.
Compared with PBS controls, all levels of protein employed in the
assays diminished the capacity of EPEC to adhere to the HEp-2 cells
(P < 0.05).
|
|
To compare the adherence-inhibition properties of the buoyant density
fractions from MUC2- or MUC3-enriched HT-29 cells, equal amounts (100 µg Lowry protein/well) of each fraction were incubated with 2.5 × 106 E2348/69/well. The
evidence for enhanced expression of specific mucin proteins (increased
mRNA expression levels) is necessarily indirect at this point because
antibodies are not available that discriminate MUC2 and MUC3 production
in spent culture media. We have previously found that, using these
techniques, mucins are contained in the most dense fractions (26). This
is a finding that would be compatible with the broad range of molecular
weights that MUC2 and MUC3 mRNA have been found to have on agarose
electrophoresis (10, 12, 13, 25). Similar to these previous findings, the least-dense fractions (fractions
4-8) derived from MUC2-enriched HT-29 cells did
not inhibit EPEC binding; however, the more dense fractions (expected
to contain mucins) inhibited EPEC binding (Fig.
8A).
Similarly, the least-dense fractions (fraction
7-8) from supernatants of MUC3-enriched HT-29
cells that would not be expected to contain mucins (24, 26) did not
inhibit the binding of EPEC to HEp-2 cells (Fig.
8B), but the more dense fractions did inhibit binding. Also, MUC3-enriched buoyant density fractions decreased E2348/69 binding to a greater extent than the corresponding MUC2-enriched fractions (Table 1).

View larger version (30K):
[in this window]
[in a new window]

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 8.
Inhibition of EPEC adherence to HEp-2 cells by exogenous mucin. To each
well of HEp-2 cells, 2.5 × 106 CFU/well of EPEC E2348/69 were
added with an equal protein content (100 µg) from buoyant density
fractions of MUC2-enriched spent HT-29 cell media
(A) or MUC3-enriched HT-29 cell
spent media (B). After a 3-h
incubation, EPEC adherent to HEp-2 cells were quantified by determining
CFU. Compared with PBS controls, binding of EPEC with high-density
fractions derived from MUC2- and MUC3-enriched media (F1-F3)
inhibited EPEC binding to HEp-2 cells
(P < 0.05). In contrast, low-density
fractions (F7 and F8) derived from both media did not inhibit EPEC
binding compared with controls (P > 0.05). Middle-density fractions (F4-F6) derived from MUC3-enriched
mucin fractions inhibited EPEC binding
(P < 0.05), whereas middle-density
fractions from MUC2-enriched fractions did not
(P > 0.05). Results are shown as
means ± SE of 4 triplicate experiments.
* P < 0.05 compared with
control.
|
|
Alterations in MUC2 and MUC3 mucin mRNA
levels. Levels of MUC2 (Fig.
9A) and
MUC3 (Fig. 9B) mRNA, normalized to
28S ribosomal RNA, were increased upon incubation of the HT-29 cells
with an inoculum of probiotic bacteria that almost completely inhibited EPEC adherence. Compared with controls, relative expression levels of
MUC2 mRNA for HT-29 cells grown in glucose culture medium incubated with 4.5 × 1010
L. plantarum 299v were increased (228 ± 48%, P < 0.05), but MUC2 expression was not altered upon coculture with 4.5 × 106 EPEC E2348/69 (146 ± 18%,
P > 0.05; Fig.
9A). Similarly, for HT-29 cells
grown in glucose-free galactose-containing culture medium (MUC3
induced), there was increased MUC3 expression for HT-29 cells incubated
with L. plantarum 299v (207 ± 48%, P < 0.05) compared
with controls, but expression by HT-29 cells incubated with EPEC
E2348/69 (79 ± 24%, P > 0.05)
was no different from controls (Fig.
9B).

View larger version (33K):
[in this window]
[in a new window]

View larger version (29K):
[in this window]
[in a new window]
|
Fig. 9.
Relative MUC2 and MUC3 mRNA expression levels after bacterial
incubation. HT-29 cells were grown in glucose-containing media
(A) or glucose-free,
galactose-containing media (B).
Bacteria were added to flasks in equivalent numbers to bacteria added
to wells of the 12-well plates, based on a surface area of
105 EPEC E2348/69 or
109 L. plantarum. After a 1-h incubation, total RNA was
collected and separated by electrophoresis on a 1.2%
agarose/formaldehyde gel before being transferred to a nitrocellulose
membrane. Hybridization using random-primed
32P-labeled MUC2 and MUC3 cDNA
probes of the respective tandem repeats was performed. Relative mucin
mRNA levels were quantified from area integration of Phosphor screen
autoradiography, normalized by dividing the densitometer values of
photographic negatives for corresponding 28S RNA levels on agarose
gels. MUC2 expression of HT-29 cells grown in glucose-containing media
was increased in the presence of L. plantarum 299v (P < 0.05) but not with EPEC E2348/69 (P > 0.05) compared with controls with PBS alone
(A). Similarly, MUC3 expression of
HT-29 cells grown in glucose-free, galactose-containing media was
increased in the presence of L. plantarum 299v (P < 0.05) but not with EPEC E2348/69 (P > 0.05)
compared with controls with PBS alone
(B). Results are shown as means ± SE from 5 separate experiments.
* P < 0.05 vs. control.
|
|
Experiments (n = 6) using EHEC
serotype O157:H7 yielded results similar to EPEC. Compared with
controls, relative expression levels of MUC2 mRNA from HT-29 cells
grown in glucose culture medium (MUC2 induced) were greater for cells
incubated with L. plantarum 299v (251 ± 49% of control, P < 0.05) but
not with E. coli O157:H7 (187 ± 43% of control, P > 0.05).
Similarly, MUC3 mRNA expression in HT-29 cells grown in galactose
culture medium was increased with incubation of L. plantarum 299v (204 ± 50% of control,
P < 0.05) but not with incubation of
O157:H7 (116 ± 15% of control, P > 0.05) compared with controls.
In separate experiments, the addition of 4.5 × 106 EPEC strain E2348/69 did not
lead to the detection of greater MUC2 mRNA levels above cells without
bacteria added (P > 0.05). However, an inoculum of 4.5 × 1010
EPEC strain E2348/69 incubated for 1 h with HT-29 cells grown in a
glucose medium showed increased MUC2 mRNA expression over both control
cells and HT-29 cells with 4.5 × 106 EPEC
(P < 0.05) added per flask as shown
in Fig. 10. Similar findings with
4.5 × 1010 EPEC
strain E2348/69 were also found with MUC3 expression in galactose-grown
HT-29 cells. The addition of sterile supernatants from
L. plantarum 299v also led to
increased MUC2 expression (145 ± 10% of control,
P < 0.01, n = 6) and MUC3 expression (149 ± 8% of control, P < 0.01, n = 6) in galactose-grown HT-29 cells (MUC3 enriched).

View larger version (37K):
[in this window]
[in a new window]
|
Fig. 10.
Relative MUC2 mucin mRNA expression levels after EPEC strain E2348/69
incubation. Various amounts of bacteria were added to glucose-grown
HT-29 cells. After a 1-h incubation, total RNA was collected,
separated, and hybridized with a MUC2 cDNA probe as previously
described. Results of the MUC2 signals are area integration relative to
28S RNA levels and are expressed as percent increase above control
values. No differences in MUC2 mRNA expression levels were found for
105 EPEC/well added and controls
(P > 0.05). Increased MUC2 mRNA expression was
detected with 109 EPEC/well
compared with both 105 EPEC/well
and control levels (* P < 0.05). Results
are shown as means ± SE from 3 separate experiments.
|
|
 |
DISCUSSION |
In the current study, two probiotic
Lactobacillus strains were shown to
inhibit in vitro adherence of EPEC or EHEC to the intestinal epithelial
HT-29 cell line. Inhibition of EPEC binding to a nonintestinal
epithelial cell line (HEp-2 cells), however, did not occur during
coincubation studies with L. plantarum
strain 299v. A number of factors are important for EPEC adherence to epithelial cells, including temperature, growth phase of EPEC, and pH
conditions (17, 30). For these studies, standardized conditions for
bacterial induction of the attachment and effacement lesion in
epithelial cells included a constant number of EPEC added to each well,
constant temperatures (37°C), and sodium bicarbonate buffering
capacity in the cell growth medium. During the 3-h incubation, there
was a change in the pH conditions with the greater inocula of the
L. plantarum 299v as evidenced by
changes in the phenol red indicator in the cell growth media. However,
the observed change in pH was the same for studies with HT-29 cells and
HEp-2 cells. The pH at the end of the incubation period is within the range of pH that allows for EPEC secretion of proteins necessary for
the induction of the attachment and effacement lesions (17).
There are a number of possible explanations whereby probiotics effect
benefit to the host. In this study, our experimentation showed that a
direct antimicrobial effect of L. plantarum strain 299v against EPEC is unlikely. The
inhibition of EPEC binding to the intestinal cell line (HT-29 cells)
and lack of inhibition of binding to a nonintestinal epithelial cell
target (HEp-2 cells) disproves this possibility. Furthermore, direct
coincubation of the two bacterial strains did not yield diminished
numbers of viable EPEC numbers. Because EPEC strain E2348/69 can
transfer its own receptor for intimate adherence to mammalian cells
(18), interference by Lactobacillus
strains with the epithelial cell EPEC receptor on one cell line and not
the other also seems improbable.
Binding to epithelial cells is the first step for many enteric
pathogens to effect net fluid and electrolyte secretion, and so
interruption of the enteropathogen adherence to intestinal epithelial
cells could provide therapeutic benefit to the host. The process
whereby EPEC inflict their characteristic attachment and effacement on
epithelial cells is a multistage process, the first stage of which is
characterized by an initial interaction of bacteria with the enterocyte
layer. This initial attachment is thought to be mediated by the
bundle-forming pilus, but other virulence factors may be involved (29).
The increased intestinal mucin production elicited by probiotics could
prevent the attachment of enteropathogens through steric hindrance or
through the effects of greater competitive inhibition for attachment
sites on mucins mimicking epithelial cell bacterial attachment sites.
Specificity in the capability of mucins from different regions of the
intestinal tract to inhibit EPEC in vitro adherence (22) and
identification of an EHEC O157:H7 mucin receptor (32) would favor the
latter explanation.
We propose that probiotic agents, such as L. plantarum 299v, which are able to bind to epithelial
cells in vitro and colonize the intestinal tract in vivo (15), induce
epithelial cells to secrete mucins that diminish enteric pathogens
binding to mucosal epithelial cells. Bacterial exoproducts from
Pseudomonas aeruginosa have been
reported to induce MUC2 mucin expression (21). Lipopolysaccharide produced from the gram-negative bacillus would not be relevant for
gram-positive bacteria such as
Lactobacilli. The effect of sterile
supernatants suggests that other cell wall determinants or secreted
products may be responsible for the increased intestinal mucin gene
expression in epithelial cells. Whether this ability to diminish
adherence is initiated through the binding of the probiotic agent to
intestinal epithelial cells in vivo and/or together with a bacterial
cell determinant or secreted bacterial product remains to be
determined, but the effect of sterile supernatants suggests multiple
possibilities are of importance.
In addition to eliciting chemokines that activate the mucosal immune
responses over a period of days, intestinal epithelial cells have fast
protective responses as part of their innate defenses, which include
the elaboration of mucins within minutes or hours of insult. Mucins
isolated from intestinal tracts of animals inhibit in vitro adherence
of both animal and human EPEC strains and bind to human EHEC (7, 26,
37). HT-29 cells grown in regular glucose culture medium express
significant levels of MUC2 mucin mRNA and can be induced to express
high levels of MUC3 mucins by different culture conditions. In
contrast, we show here that HEp-2 cells have minimal MUC2 mucin mRNA
expression and no MUC3 mucin expression, and increased expression by
incubation of L. plantarum 299v was
not demonstrated. MUC2 and MUC3 mucin fractions isolated from HT-29
cell spent culture media and added exogenously to HEp-2 cells were
capable of inhibiting EPEC binding. HT-29 cells are not the only
intestinal epithelial cell line for which coincubation with probiotics
led to diminished EPEC adherence. In previous studies by Bernet et al.
(1), a Lactobacillus acidophilus strain was shown to inhibit EPEC binding to another intestinal cell
line, Caco-2 cells. We previously showed that the Caco-2 cell line can
express significant levels of MUC2, MUC3, and MUC4 mucin mRNA (13).
Thus increased intestinal mucin production may also explain the
previously reported effects of probiotics on Caco-2 cells.
MUC3-enriched mucin fractions inhibited EPEC binding to a greater
extent than MUC2 mucins. This may be an important biological attribute
of intestinal mucins, since cellular expression of MUC3 mucin is
greater then MUC2 in the small intestine (12, 38). Discharges from
goblet cells, which contain high levels of MUC2 mucins, are increased
during inflammation of the intestinal tract and may be a secondary line
of defense in addition to intestinal columnar cell mucin production.
There are other possibilities for the differences in the capacities of
MUC2 and MUC3 mucins to inhibit EPEC adherence. For example, HT-29
cells grown in galactose culture medium alter expression of MUC3
intestinal mucins and carbohydrate antigen expression on secreted and
cell surface-associated mucins (23). Future determination of molecular
interactions between EPEC and mucins may provide insight into this phenomenon.
In addition to composition, quality and quantity are also factors of
intestinal mucins that may contribute to pathogen-mucin interactions
(7). There are reduced numbers of goblet cells in inflammatory lesions
of the bowel, such as Crohn's disease and ulcerative colitis. Mucins
from inflamed colons have decreased functional capacity to bind
proinflammatory molecules (6) and to inhibit bacterial binding (24).
However, administration of L. plantarum 299v is effective in reducing enterocolitis
in an animal model of intestinal inflammation (27). The dose of
L. plantarum 299v used in our studies
was similar to that used in previous studies that showed almost
complete inhibition of EPEC and EHEC binding to HT-29 cells. In
addition, the same dose of L. plantarum 299v increased expression of MUC2 mucin and
MUC3 mucin mRNA after a 1-h coincubation (Fig. 9). A subagglutinating concentration of 105 EPEC used in
our inhibition studies did not induce upregulation in the expression of
MUC2 or MUC3 intestinal mucins; however, a greater inoculum was capable
of this phenomenon. Infection of another intestinal adenocarcinoma cell
line, T84 cells, with EPEC leads to the activation of NF-
B (34).
P. aeruginosa-activated NF-
B has
been demonstrated to bind to a
B site in the 5'-flanking region of the MUC2 gene and activates MUC2 mucin transcription in the
HM3 colon epithelial cell line (21). How the
Lactobacilli may cause increased mucin
gene transcription remains to be determined.
The benefits of probiotics mediated through intestinal mucin
upregulation may have broader applicability than only for bacterial enteropathogens. For instance, Yolken at al. (40) showed that intestinal mucins inhibit rotavirus replication. Because probiotics can
increase expression of intestinal mucins, the reduction of both
symptoms and fecal shedding after the onset of acute rotavirus gastroenteritis in children after administration of probiotic agents
(9, 31, 36) may be by way of this mechanism. However, our in vitro
studies did not show that EPEC could be dislodged by probiotics. Thus
whether enhancement of innate defense mechanisms of intestinal
epithelial cells such as mucin production is preventative or
therapeutic for any specific intestinal infection remains to be determined.
 |
ACKNOWLEDGEMENTS |
This work is supported by National Institute of Diabetes and
Digestive and Kidney Diseases Grant DK-02205 and in part from a
unrestricted grant from ConAgra.
 |
FOOTNOTES |
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: D. R. Mack,
985160 Nebraska Medical Center, Omaha, NE 68198-5160.
Received 1 July 1998; accepted in final form 21 December 1998.
 |
REFERENCES |
1.
Bernet, M. F.,
D. Brassart,
J. R. Neeser,
and
A. L. Servin.
Lactobacillus acidophilus LA 1 binds to cultured human intestinal cell lines and inhibits cell attachment and cell invasion by enterovirulent bacteria.
Gut
35:
483-489,
1994[Abstract].
2.
Brasaemle, S. R.,
and
A. D. Attie.
Microelisa reader quantification of fixed, stained, solubilized cells in microtitre dishes.
Biotechniques
6:
418-419,
1988[Medline].
3.
Chang, S. K.,
A. F. Dohrman,
C. B. Basbaum,
S. B. Ho,
T. Tsuda,
N. W. Toribara,
J. R. Gum,
and
Y. S. Kim.
Localization of mucin (MUC2 and MUC3) messenger RNA and peptide expression in human normal intestine and colon cancer.
Gastroenterology
107:
28-36,
1994[Medline].
4.
Dytoc, M.,
R. Soni,
F. Cockerill,
J. de Azavedo,
M. Louie,
J. Brunton,
and
P. Sherman.
Multiple determinants of verocytotoxin-producing Escherichia coli O157: H7 attachment-effacement.
Infect. Immun.
61:
3382-3391,
1993[Abstract].
5.
Elmer, G. W.,
C. M. Surawicz,
and
L. V. McFarland.
Biotherapeutic agents. A neglected modality for the treatment and prevention of selected intestinal and vaginal infections.
JAMA
275:
870-876,
1996[Abstract].
6.
Ferry, D. M.,
T. J. Butt,
M. F. Broom,
J. Hunter,
and
V. S. Chadwick.
Bacterial chemotactic oligopeptides and the intestinal mucosal barrier.
Gastroenterology
97:
61-67,
1989[Medline].
7.
Forstner, J. F.,
and
G. G. Forstner.
Gastrointestinal mucus.
In: Physiology of the Gastrointestinal Tract (3rd ed.), edited by L. R. Johnson. New York: Raven, 1994, p. 1255-1283.
8.
Gorbach, S. L.
The discovery of Lactobacillus GG.
Nutrition Today Suppl.
31:
2S-4S,
1996.
9.
Guarino, A.,
R. B. Canani,
M. I. Spagnuolo,
F. Albano,
and
L. Di Benedetto.
Oral bacterial therapy reduces the duration of symptoms and of viral excretion in children with mild diarrhea.
J. Pediatr. Gastroenterol. Nutr.
25:
516-519,
1997[Medline].
10.
Gum, J. R., Jr.
Mucins; their structure and biology.
Biochem. Soc. Trans.
23:
795-799,
1995[Medline].
11.
Gum, J. R., Jr.,
J. J. L. Ho,
W. S. Pratt,
J. W. Hicks,
A. S. Hill,
L. E. Vinall,
A. M. Roberton,
D. M. Swallow,
and
Y. S. Kim.
MUC3 human intestinal mucin. Analysis of gene structure, the carboxyl terminus, and a novel upstream repetitive region.
J. Biol. Chem.
272:
26678-26686,
1997[Abstract/Free Full Text].
12.
Ho, S. B.,
G. A. Niehans,
C. Lyftogt,
P. S. Yan,
D. L. Cherwitz,
E. T. Gum,
R. Dahiya,
and
Y. S. Kim.
Heterogeneity of mucin gene expression in normal and neoplastic tissues.
Cancer Res.
53:
641-651,
1993[Abstract].
13.
Hollingsworth, M. A.,
J. M. Strawhecker,
T. C. Caffrey,
and
D. R. Mack.
Expression of MUC1, MUC2, MUC3 and MUC4 mucin mRNAs in human pancreatic and intestinal tumor cell lines.
Int. J. Cancer
57:
198-203,
1994[Medline].
14.
Jerse, A. E.,
K. G. Gicquelais,
and
J. B. Kaper.
Plasmid and chromosomal elements involved in the pathogenesis of attaching and effacing Escherichia coli.
Infect. Immun.
59:
3869-3875,
1991[Medline].
15.
Johansson, M. L.,
G. Molin,
B. Jeppsson,
S. Nobaek,
S. Ahrne,
and
S. Bengmark.
Administration of different Lactobacillus strains in fermented oatmeal soap: in vivo colonization of human intestinal mucosa and effect on the indigenous flora.
Appl. Environ. Microbiol.
59:
15-20,
1993[Abstract].
16.
Jung, H. C.,
L. Eckmann,
S.-K. Yang,
A. Panja,
J. Fierer,
E. Morzycka-Wroblewska,
and
M. F. Kagnoff.
A distinct array of proinflammatory cytokines is expressed in human colon epithelial cells in response to bacterial invasion.
J. Clin. Invest.
95:
55-65,
1995[Medline].
17.
Kenny, B.,
A. Abe,
M. Stein,
and
B. B. Finlay.
Enteropathogenic Escherichia coli protein secretion is induced in response to conditions similar to those in the gastrointestinal tract.
Infect. Immun.
65:
2606-2612,
1997[Abstract].
18.
Kenny, B.,
R. DeVinney,
M. Stein,
D. J. Reinscheid,
E. A. Frey,
and
B. B. Finlay.
Enteropathogenic E. coli (EPEC) transfers its receptor for intimate adherence into mammalian cells.
Cell
91:
511-520,
1997[Medline].
19.
Klapproth, J.-M.,
M. S. Donnenberg,
J. M. Abraham,
and
S. P. James.
Products of enteropathogenic Escherichia coli inhibit lymphokine production by gastrointestinal lymphocytes.
Am. J. Physiol.
271 (Gastrointest. Liver Physiol. 34):
G841-G848,
1996[Abstract/Free Full Text].
20.
Levine, M. M.,
J. P. Nataro,
H. Karch,
M. M. Baldini,
J. B. Kaper,
R. E. Black,
M. L. Clements,
and
A. D. O Brien.
The diarrheal response of humans to some classic serotypes of enteropathogenic Escherichia coli is dependent on a plasmid encoding an enteroadhesiveness factor.
J. Infect. Dis.
152:
550-559,
1985[Medline].
21.
Li, J.-D.,
W. Feng,
M. Gallup,
J.-H. Kim,
J. Gum,
Y. Kim,
and
C. Basbaum.
Activation of NF-
B via a Src-dependent Ras-MAPK-pp90rsk pathway is required for Pseudomonas aeruginosa-induced mucin overproduction in epithelial cells.
Proc. Natl. Acad. Sci. USA
95:
5718-5723,
1998[Abstract/Free Full Text].
22.
Mack, D. R.,
and
P. L. Blain-Nelson.
Disparate in vitro inhibition of adhesion of enteropathogenic Escherichia coli RDEC-1 by mucins isolated from various regions of the intestinal tract.
Pediatr. Res.
37:
75-80,
1995[Abstract].
23.
Mack, D. R., P.-W. Cheng, F. Perini, S. Wei, and
M. A. Hollingsworth. Altered expression of sialylated
carbohydrate antigens in HT29 colonic carcinoma cells.
Glycoconjugate J. In
press.
24.
Mack, D. R.,
T. S. Gaginella,
and
P. M. Sherman.
Effect of colonic inflammation on mucin inhibition of Escherichia coli RDEC-1 binding in vitro.
Gastroenterology
102:
1199-1211,
1992[Medline].
25.
Mack, D. R.,
and
M. A. Hollingsworth.
Alteration in expression of MUC2 and MUC3 mRNA levels in HT29 colonic carcinoma cells.
Biochem. Biophys. Res. Commun.
199:
1012-1018,
1994[Medline].
26.
Mack, D. R.,
and
P. M. Sherman.
Mucin isolated from rabbit colon inhibits in vitro binding of Escherichia coli RDEC-1.
Infect. Immun.
59:
1015-1023,
1991[Medline].
27.
Mao, Y.,
S. Nobaek,
B. Kasravi,
D. Adawi,
U. Stenram,
G. Molin,
and
B. Jeppsson.
The effects of Lactobacillus strains and oat fiber on methotrexate-induced enterocolitis in rats.
Gastroenterology
111:
334-344,
1996[Medline].
28.
McDaniel, T. K.,
K. G. Jarvis,
M. S. Donnenberg,
and
J. B. Kaper.
A genetic locus of enterocyte effacement conserved among diverse enterobacterial pathogens.
Proc. Natl. Acad. Sci. USA
92:
1664-1668,
1995[Abstract].
29.
Nataro, J. P.,
and
J. B. Kaper.
Diarrheagenic Escherichia coli.
Clin. Microbiol. Rev.
11:
142-201,
1998[Abstract/Free Full Text].
30.
Rosenshine, I.,
S. Ruschkowski,
and
B. B. Finlay.
Expression of attachment/effacing activity by enteropathogenic Escherichia coli depends on growth phase, temperature and protein synthesis upon contact with epithelial cells.
Infect. Immun.
64:
966-973,
1996[Abstract].
31.
Saaverda, J. M.,
N. A. Bauman,
I. Oung,
J. A. Perman,
and
R. H. Yolken.
Feeding of Bifidobacterium bifidum and Streptococcus thermophilus to infants in hospital for prevention of diarrhoea and shedding of rotavirus.
Lancet
344:
1046-1049,
1994[Medline].
32.
Sajjan, S. U.,
and
J. F. Forstner.
Role of the putative link glycopeptide of intestinal mucin in binding of piliated Escherichia coli serotype O157:H9 strain CL49.
Infect. Immun.
58:
868-873,
1990[Medline].
33.
Savkovic, S. D.,
A. Koutsouris,
and
G. Hecht.
Attachment of a noninvasive enteric pathogen, enteropathogenic Escherichia coli, to cultured human intestinal epithelial monolayers induces transmigration of neutrophils.
Infect. Immun.
64:
4480-4487,
1996[Abstract].
34.
Savkovic, S. D.,
A. Koutsouris,
and
G. Hecht.
Activation of NF-
B in intestinal epithelial cells by enteropathogenic Escherichia coli.
Am. J. Physiol.
273 (Cell Physiol. 42):
C1160-C1167,
1997[Medline].
35.
Saxelin, M.,
S. Elo,
and
S. Salminen.
Dose response colonization of feces after oral administration of Lactobacillus casei strain GG.
Microbiol. Ecol. Health Dis.
4:
209-214,
1991.
36.
Shornikova, A.-V.,
I. A. Casas,
E. Isolauri,
H. Mykkanen,
and
T. Vesikari.
Lactobacillus reuteri as a therapeutic agent in acute diarrhea in young children.
J. Pediatr. Gastroenterol. Nutr.
24:
399-404,
1997[Medline].
37.
Smith, C. J.,
J. B. Kaper,
and
D. R. Mack.
Intestinal mucin inhibits adhesion of human enteropathogenic Escherichia coli to HEp-2 cells.
J. Pediatr. Gastroenterol. Nutr.
21:
269-276,
1995[Medline].
38.
Van Klinken, B. J.,
K. M. A. J. Tytgat,
H. A. Buller,
A. W. C. Einerhand,
and
J. Dekker.
Biosynthesis of intestinal mucins: MUC1, MUC2, MUC3 and more.
Biochem. Soc. Trans.
23:
814-818,
1995[Medline].
39.
Yang, S.-K.,
L. Eckmann,
A. Panja,
and
M. F. Kagnoff.
Differential and regulated expression of C-X-C, C-C, and C-chemokines by human colon epithelial cells.
Gastroenterology
113:
1214-1223,
1997[Medline].
40.
Yolken, R. H.,
C. Ojeh,
I. A. Khatri,
U. Sajjan,
and
J. F. Forstner.
Intestinal mucins inhibit rotavirus replication in an oligosaccharide-dependent manner.
J. Infect. Dis.
169:
1002-1006,
1994[Medline].
Am J Physiol Gastroint Liver Physiol 276(4):G941-G950
0002-9513/99 $5.00
Copyright © 1999 the American Physiological Society