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Applied and Environmental Microbiology, March 2000, p. 1152-1157, Vol. 66, No. 3
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Antagonistic Activity of Lactobacillus
acidophilus LB against Intracellular Salmonella
enterica Serovar Typhimurium Infecting Human Enterocyte-Like
Caco-2/TC-7 Cells
Marie-Hélène
Coconnier,
Vanessa
Liévin,
Mathie
Lorrot, and
Alain L.
Servin*
Pathogènes et Fonctions des Cellules
Epithéliales Polarisées, Institut National de la
Santé et de la Recherche Médicale, Unité 510, Faculté de Pharmacie, Université Paris XI, F-92296
Châtenay-Malabry, France
Received 22 November 1999/Accepted 16 December 1999
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ABSTRACT |
To gain further insight into the mechanism by which lactobacilli
develop antimicrobial activity, we have examined how
Lactobacillus acidophilus LB inhibits the promoted cellular
injuries and intracellular lifestyle of Salmonella enterica
serovar Typhimurium SL1344 infecting the cultured, fully differentiated
human intestinal cell line Caco-2/TC-7. We showed that the spent
culture supernatant of strain LB (LB-SCS) decreases the number of
apical serovar Typhimurium-induced F-actin rearrangements in infected
cells. LB-SCS treatment efficiently decreased transcellular passage of
S. enterica serovar Typhimurium. Moreover, LB-SCS treatment
inhibited intracellular growth of serovar Typhimurium, since treated
intracellular bacteria displayed a small, rounded morphology resembling
that of resting bacteria. We also showed that LB-SCS treatment inhibits
adhesion-dependent serovar Typhimurium-induced interleukin-8 production.
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INTRODUCTION |
Salmonella spp. are the
etiologic agents of a variety of diseases globally defined as
salmonellosis. Infections range in severity from self-limiting
gastroenteritis to life-threatening systemic enteric fevers to
septicemia. Studies have shown that salmonellae initiate infection by
invading and multiplying within epithelial intestinal cells and
Peyer's patches. Salmonella enterica serovar Typhimurium
interacts with microvilli of the intestinal brush border (9,
16), inducing the formation of surface appendages (17)
and promoting the synthesis of bacterial proteins (10). During the entry process, protrusion of the host cell membrane occurs,
and the bacteria are completely surrounded by "splashes" of
membrane, indicative of macropinocytosis (11). Changes in the distribution of cytoskeletal components, resulting from signaling (for a review, see reference 13), have been reported
during Salmonella invasion (11, 12, 15).
Cytoskeletal rearrangements and membrane ruffling involve the small
guanosine triphosphate-binding protein CDC42Hs (3). Invasion
of M cells in vivo and of epithelial cells and macrophages in vitro
requires a type III secretion system encoded on the
Salmonella SPI1 pathogenicity island located at centisome 63 (7). After entry, bacteria reside in vacuoles and obtain
nutrients from the host eucaryotic cells (14), allowing intracellular growth of the microorganisms (20).
We have documented the antagonistic activity, in vitro and in vivo, of
Lactobacillus acidophilus LB against enterovirulent bacteria
involved in gastrointestinal disorders. For example, this strain causes
a significant inhibition of enteropathogens' association with and
entry into cultured human intestinal cells (4-6). Moreover,
we have recently provided evidence that strain LB secretes a
heat-stable antimicrobial compound(s) different from lactic acid
(5). The aim of this study was to gain further insight into
the antagonistic activity of L. acidophilus LB against S. enterica serovar Typhimurium, in particular against
bacteria localized within cultured human enterocyte-like Caco-2/TC-7
cells, a model of the mature enterocyte of the small intestine
(23).
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MATERIALS AND METHODS |
Bacterial strains and growth conditions.
S.
enterica serovar Typhimurium strain SL 1344 (9) was
used as the indicator strain. Bacteria were cultured in Luria broth (Difco Laboratories, Detroit, Mich.) at 37°C.
L. acidophilus LB was isolated from a human stool specimen
(Lacteol Laboratory, Houdan, France). LB bacteria were grown in DeMan,
Rogosa, Sharpe (MRS) broth (Biokar Diagnostic, Beauvais, France) for
18 h at 37°C. Spent culture supernatant of LB (LB-SCS) was
obtained by centrifugation of the culture at 10,000 × g and 4°C for 30 min. Centrifuged LB-SCS was passed through a
sterile 0.22-µm-pore-size filter unit (Millex GS; Millipore,
Molsheim, France). Filtered LB-SCS was assessed for the absence of
bacteria by plating on tryptic soy agar. A concentrated (twofold)
suspension of the LB-SCS was obtained by freeze-drying. The pH values
of different LB-SCS preparations ranged from 5 to 4.5. As a control, MRS was adjusted to pH 4.5 with HCl (MRS-HCl) as previously described (2, 4-6). As a second control, MRS was adjusted to pH 4.5 with DL-lactic acid (MRS-LA) (final concentration, 100 mM).
Caco-2/TC-7 cell culture.
The TC7 clone (Caco-2/TC-7)
(1), established from the parental Caco-2 cell line
(23), was used. Cells were routinely grown in Dulbecco
modified Eagle's medium (DMEM) (25 mM glucose) (Life Technologies,
Cergy, France), supplemented with 20% heat-inactivated (30 min,
56°C) fetal calf serum (Boehringer, Mannheim, Germany) and 1%
nonessential amino acids (Life Technologies) as previously described
(2, 4-6). For maintenance purposes, cells were passaged weekly, using 0.02% trypsin in Ca2+- and
Mg2+-free phosphate-buffered saline (PBS) containing 3 mM
EDTA. Experiments and maintenance of the cells were carried out at
37°C in a 10% CO2-90% air atmosphere. The culture
medium was changed daily. For assays of S. enterica serovar
Typhimurium infection, Caco-2/TC-7 cells were used at postconfluence
after 15 days of culture (i.e., they were fully differentiated cells).
Determination of penetration of S. enterica serovar
Typhimurium into Caco-2/TC-7 cells.
For Caco-2/TC-7 cell monolayer
infection, serovar Typhimurium strain SL1344 was cultured at 37°C for
18 h in Luria broth. A cell infection assay was conducted as
previously reported (2, 4, 5). Briefly, prior to infection,
the Caco-2/TC-7 monolayers, prepared in 24-well tissue culture plates
(TPP) (ATGC, Paris, France), were washed twice with PBS. Bacteria were
suspended in the culture medium, and 1 ml of this suspension was added
to each well of the tissue culture plate (5 × 107
CFU/well). The plates were incubated at 37°C in an atmosphere of 10%
CO2-90% air for different infection time periods and then were washed three times with sterile PBS.
Internalization of serovar Typhimurium organisms was determined by
quantitative measurement of numbers of bacteria located
within the
infected monolayers, using the aminoglycoside antibiotic
assay (
4,
5). After incubation, monolayers were washed twice
with sterile
PBS and then incubated for 60 min in medium containing
50 µg of
gentamicin/ml. Bacteria that adhered to the Caco-2/TC-7
brush border
were rapidly killed, whereas those located within
Caco-2/TC-7 cells
survived. The monolayer was washed with PBS
and lysed with sterilized
H
2O. To determine the number of viable
intracellular
bacteria, appropriate dilutions were plated on tryptic
soy agar and
bacterial colony counts were
performed.
For determination of penetration by
S. enterica serovar
Typhimurium into Caco-2/TC-7 cells, the latter were grown on
1-cm
2 filters mounted in a culture chamber (Costar culture
plate inserts;
3-µm pore size) (10
5 cells per chamber),
which delineates an apical (luminal) and
a basolateral (serosal)
reservoir (
18). Bacteria (5 × 10
7
CFU/well) were inoculated onto the apical surface. After incubation,
monolayers were washed twice with sterile PBS and then incubated
for 60 min in medium containing 50 µg of gentamicin/ml, which
kills only the
extracellular bacteria. The numbers of bacteria
in the basolateral
medium were monitored over
time.
Each assay was conducted in triplicate with three successive passages
of Caco-2/TC-7
cells.
Antagonistic activity against serovar Typhimurium infection in
Caco-2/TC-7 cells.
The activity of LB-SCS against S. enterica serovar Typhimurium SL1344-induced cell alterations in
vitro was determined under two sets of experimental conditions. In one
experiment, pretreatment of SL1344 with LB-SCS was carried out for
1 h at 37°C before cell infection was conducted. After
centrifugation (5,500 × g, 10 min, 4°C), the
bacteria were washed with PBS and resuspended in the Caco-2/TC-7 cell
culture medium for the cell infection process (5 × 107 CFU/ml).
In the other experiment, Caco-2/TC-7 cells, grown on filters mounted in
a culture chamber, were infected with
S. enterica serovar
Typhimurium SL1344 (5 × 10
7 CFU/ml, 1 h, 37°C)
to determine the activity of LB-SCS against
this intracellular
pathogen. After infection, monolayers were
washed twice with sterile
PBS and then extracellular salmonellae
were killed by treatment with
gentamicin (50 µg/ml, 1 h, 37°C).
Monolayers were washed twice
with sterile PBS and subjected to
LB-SCS (twofold concentrated)
treatment for 1 h at 37°C. In all
experiments, determinations of
the viable cell-associated and
intracellular bacteria and of
penetration (sorting in basolateral
medium) of serovar Typhimurium were
conducted as described
above.
Each assay was conducted in triplicate with three successive passages
of Caco-2/TC-7
cells.
Immunofluorescence microscopy.
Monolayers of Caco-2/TC-7
cells were prepared on glass coverslips which were placed in 24-well
tissue culture plates. After infection, cell preparations were fixed
for 10 min at room temperature in PBS-3.5% paraformaldehyde.
Determination of F-actin cytoskeletal cell rearrangements was conducted
by direct immunofluorescence microscopy, using fluorescein-phalloidin
(Molecular Probes, Junction City, Oreg.). The coverslips were incubated
with PBS-0.2% Triton X-100 for 4 min before incubation with
fluorescein-phalloidin for 45 min at 22°C, after which the coverslips
were washed three times with PBS.
For examination of intracellular
S. enterica serovar
Typhimurium after Caco-2/TC-7 cell infection (5 × 10
7
CFU/ml), indirect immunofluorescence microscopy was performed
with a
polyclonal antibody directed against the
Salmonella O
antigen
(diluted 1:100 in PBS) (Institut Pasteur, Paris, France).
Coverslips
were incubated with PBS-0.2% Triton X-100 for 4 min before
application
of the primary antibody, and after a 45-min incubation at
22°C,
the coverslips were washed three times with PBS. Then a
fluorescein
isothiocyanate-conjugated secondary antibody was added, and
after
another 45-min incubation at 22°C the coverslips were again
washed
three times with
PBS.
Specimens were examined by epifluorescence microscopy, using an
epifluorescence-equipped Leitz Aristoplan microscope coupled
to an
Image Analyzer Visiolab 1000 (Biocom, Les Ullis, France).
All
photographs were taken on Kodak film (Eastman Kodak Co., Rochester,
N.Y.).
IL-8 assay.
For determination of interleukin-8 (IL-8)
levels, monolayers of Caco-2/TC-7 cells were prepared in 24-well tissue
culture plates. For some experiments, serovar Typhimurium bacteria were treated with LB-SCS for 1 h at 37°C prior to monolayer infection (5 × 107 CFU/well). Under the other set of
conditions, Caco-2/TC-7 cells were apically infected with S. enterica serovar Typhimurium (5 × 107 CFU/well)
for 1 h and then treated for 1 h with LB-SCS. For this set of
experimental conditions, after removal of the culture medium the cells
were further incubated for 4 h in DMEM containing 50 µg of
gentamicin/ml. Under all conditions, the culture medium in which
cytokine levels were determined was first centrifuged for 20 min at
12,000 × g to pellet residual bacteria and cells. The
IL-8 concentration was determined with a human IL-8 immunoassay kit
(Diaclone Research Biotest, Buc, France). In a preliminary experiment,
we determined that LB-SCS does not interfere with the IL-8 immunoassays.
Data analysis.
Results are expressed as means ± standard errors of the means (SEM). For statistical comparisons,
Student's t test was performed.
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RESULTS |
LB-SCS inhibits the intracellular lifestyle of S. enterica serovar Typhimurium SL1344 in Caco-2/TC-7 cells.
When S. enterica serovar Typhimurium SL1344 (5 × 107 CFU/ml) apically infected Caco-2/TC-7 cells, it
efficiently entered the cells by 1 h postinfection
(5 × 106 CFU/ml; intracellular). Transcellular passage
of serovar Typhimurium in Caco-2/TC-7 cells has been recently
documented, and there is evidence of a basolateral sorting of S. enterica serovar Typhimurium SL1344 after apical infection
(18). We observed here that in Caco-2/TC-7 cells infected
with SL1344 for 1 h, bacteria, penetrating the cell monolayer
through the apical domain, appeared in the basolateral medium by 8 h postinfection (Fig. 1). The activity of
LB-SCS against the transcellular passage of serovar Typhimurium was
examined. A highly significant decrease in basolateral sorting of
viable serovar Typhimurium organisms was observed after the cell
monolayers that had been infected with SL1344 for 1 h were treated
with LB-SCS for 1 h. Two MRS controls were used. The first one was
MRS adjusted to pH 4.5 with HCl (MRS-HCl) as previously described
(2, 4-6). Since lactic acid was present in LB-SCS (5), the second control was MRS adjusted to pH 4.5 with
DL-lactic acid (MRS-LA) (final concentration, 100 mM). As
disclosed in Fig. 1, both the MRS-HCl and MRS-LA controls were
inactive.
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FIG. 1.
Effect of LB-SCS treatment on penetration of S. enterica serovar Typhimurium SL1344 through Caco-2/TC-7 cell
monolayers. Caco-2/TC-7 cell monolayers grown on filters mounted in a
culture chamber were inoculated with SL1344 at 5 × 107 CFU/well. At 2 h postinfection, the cells were
treated with MRS-HCl, MRS-LA, or LB-SCS for 1 h. In control and
treated cells, the numbers of bacteria in the basolateral medium were
monitored over a period of 8 h posttreatment. Each value shown is
the mean ± SEM of data from three experiments. Statistical
analysis was performed with Student's t test. There was a
highly significant difference (P < 0.001) between
control SL1344 and LB-SCS values.
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We conducted additional experiments to examine the development of
LB-SCS activity. Considering the result above, we determined
the level
of viable intracellular serovar Typhimurium, as a function
of time of
LB-SCS treatment, in Caco-2/TC-7 cells that had been
infected with
SL1344 for 2 h (Fig.
2A). As shown
in Fig.
2, the
level of viable intracellular bacteria decreased
regularly during
the 2-h period of LB-SCS treatment. For example, when
measured
at 2 h, a 3-log decrease in viable intracellular serovar
Typhimurium
was observed, whereas a 1-log decrease was observed after
treatment
with MRS-HCl or MRS-LA under the same conditions.
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FIG. 2.
Effect of LB-SCS treatment on S. enterica
serovar Typhimurium SL1344 residing within Caco-2/TC-7 cells. (A)
Caco-2/TC-7 cells that had been infected with SL144 for 2 h were
treated with LB-SCS, MRS-HCl, or MRS-LA, and the level of intracellular
bacteria was examined during the 2-h period of treatment. (B) A
monolayer of Caco-2/TC-7 cells that had been infected with SL1344 for
2 h was treated with LB-SCS, MRS-HCl, or MRS-LA for 1 h. The
level of intracellular bacteria was examined over a period of 20 h
posttreatment. Each value shown is the mean ± SEM for values from
three experiments.
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We examined how intracellular
S. enterica serovar
Typhimurium evolved after LB-SCS treatment over a long period of time
postinfection
(Fig.
2B). For this purpose, a Caco-2/TC-7 cell monolayer
that
had been infected with SL1344 for 2 h was treated for 1 h with
LB-SCS. In agreement with a recent study showing that serovar
Typhimurium did not proliferate in nonphagocytic cells (
21),
we found that
S. enterica serovar Typhimurium SL1344 did not
proliferate
in the nonphagocytic Caco-2/TC-7 cells. While examining how
the
level of intracellular bacteria evolves, we observed that a 4-log
decrease in the level of viable intracellular serovar Typhimurium
occurred during the first 3 h post-LB-SCS treatment; afterward,
the level of intracellular bacteria remained stable through 20
h
posttreatment. We observed that after a 1-h treatment with MRS-HCl
or
MRS-LA under the same conditions, only a 1.5-log decrease in
the level
of viable intracellular serovar Typhimurium occurred,
and the level
remained stable through 20 h
posttreatment.
The behavior of intracellular
S. enterica serovar
Typhimurium SL1344 in the Caco-2/TC-7 cells was examined by a detailed
microscopic
examination of infected cells, in which intracellular
bacteria
were stained with rabbit anti-serovar Typhimurium O antigen
polyclonal
antibody (Fig.
3). In control
infected cells, two bacterial morphologies
were observed in randomly
distributed cells. The bacteria were
organized into long filaments,
with division septa initiated but
not completed, and in a large
proportion of bacterial progeny
a normal rod-shaped morphology,
indicating proliferation, was
evident. In infected cells exposed to
LBS-SCS (1 h of treatment),
no bacteria were found to have organized
into long filaments or
to have significant progeny with a normal
rod-shaped morphology.
In contrast, intracellular bacteria displayed a
small, rounded
morphology resembling that of resting bacteria. In
infected cells
exposed to MRS-HCl or MRS-LA (1 h of treatment), most
progeny
of intracellular bacteria had a normal rod-shaped morphology,
but bacteria were not organized into long filaments. This resembles
one
of the serovar Typhimurium morphologies observed in nontreated
infected
cells.
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FIG. 3.
Effect of LB-SCS treatment on S. enterica
serovar Typhimurium SL1344 residing within Caco-2/TC-7 cells. Detection
of intracellular bacteria in Triton X-100-permeabilized Caco-2/TC-7
cells was conducted by indirect immunofluorescence microscopy, using a
polyclonal antibody directed against the Salmonella O
antigen. Magnifications, ×100. (A and B) Morphologies of intracellular
bacteria present in a monolayer of Caco-2/TC-7 cells infected for
2 h with SL1344. (C) Morphology of intracellular bacteria present
in Caco-2/TC-7 cells that had been preinfected with SL1344 for 2 h, then treated with LB-SCS for 1 h. (D) Morphology of
intracellular bacteria in a monolayer of Caco-2/TC-7 cells infected for
2 h with SL1344 and then treated with MRS-LA for 1 h. Identical
results were obtained with MRS-HCl treatment (data not shown).
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Activity of LB-SCS against S. enterica serovar
Typhimurium SL1344-induced apical F-actin rearrangements in Caco-2/TC-7
cells.
It is well known that in epithelial cells, invading serovar
Typhimurium SL1344 is surrounded by a large extension of the host cell
membrane, correlating with the size and extent of an F-actin-dense region (11, 12, 15). We examined F-actin distribution at the
apical domain in SL1344-infected Caco-2/TC-7 cells by
fluorescein-phalloidin labeling and epifluorescence microscopy (Fig.
4). In the uninfected control cells, the
fine, flocculated actin located centrally in the cells represents
microvillus-associated F-actin (Fig. 4A). We found dramatic changes in
the apical F-actin distribution in serovar Typhimurium SL1344-infected
Caco-2/TC-7 cells, characterized by the disappearance of the fine,
flocculated actin located centrally in the cells and the appearance of
intense localized accumulations of F-actin (Fig. 4B and C).
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FIG. 4.
Evolution of the serovar Typhimurium SL1344-induced
apical F-actin alteration in Caco-2/TC-7 cells upon LB-SCS treatment.
High-magnification micrographs showing localization of F-actin labeled
by fluorescein-phalloidin. Magnifications, ×100. (A) Control
uninfected cells. The fine flocculated actin centrally located in the
cells represents microvillus-associated F-actin. (B and C) Cells
infected for 2 h with S. enterica serovar Typhimurium
SL1344, incubated in DMEM for 1 h, and observed at 1 h (B) or
2 h (C) posttreatment. The localized dense spots of fluorescence
represent F-actin accumulation resulting from a
Salmonella-induced lesion. (D) Uninfected cells treated for
1 h with MRS-LA, showing no change in apical F-actin distribution.
(E and F) SL1344-induced localized dense F-actin accumulation in cells
1 h (E) or 2 h (F) post-MRS-LA treatment. The randomly
dispersed serovar Typhimurium-induced localized F-actin accumulations
remain present and are of the same size and intensity and size as those
of infected untreated cells. Identical results were obtained with
LB-HCl treatment (data not shown). (G) Uninfected cells treated for
1 h with MRS-HCl, showing no change in apical F-actin
distribution. (H and I) SL1344-induced localized dense F-actin
accumulation in cells 1 h (H) or 2 h (I) post-LB-SCS
treatment. The dispersed localized Salmonella-induced
F-actin accumulations were reduced in intensity at 1 h post-LB-SCS
treatment. At 2 h post-LB-SCS treatment, no F-actin accumulation
was found and the cells showed a normal F-actin distribution.
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Evolution of
S. enterica serovar Typhimurium SL1344-induced
F-actin accumulation upon MRS or LB-SCS treatment was examined
(Fig.
4
and Table
1). For this purpose, cells
that had undergone
bacterial infection for 2 h were treated with
LB-SCS for 1 h and
F-actin accumulation was examined at 1 and
2 h posttreatment (4
and 5 h postinfection, respectively)
(Fig.
4H and I). At 1 h post-LB-SCS
treatment, F-actin
accumulation remained evident but appeared
more diffuse than in control
infected cells (Fig.
4H). At 2 h
post-LB-SCS treatment, the cell
surface return to normal, since
no F-actin accumulation was observed
(Fig.
4I), and the distribution
of the apical F-actin resembled the
distribution observed in control
uninfected cells and uninfected
LB-SCS-treated cells (Fig.
4A
and G, respectively). In contrast,
treatment of the preinfected
cells with MRS-LA did not modify
SL1344-induced F-actin accumulation
at any time posttreatment (Fig.
4E
and F). To quantify our observation,
we determined F-actin accumulation
in a blinded review of about
20 random microscopic areas in nine
monolayers resulting from
three separate experiments (Table
1). In
S. enterica serovar
Typhimurium SL1344-infected cells
treated with LB-SCS, we observed
that the level of SL1344-induced
apical F-actin accumulation decreased
significantly at 4 and 5 h
postinfection compared with the control
untreated-but-infected cells.
In contrast, no change in the level
of SL1344-induced apical F-actin
accumulation was observed upon
MRS-HCl or MRS-LA treatment.
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TABLE 1.
Evolution of S. enterica serovar Typhimurium
SL1344-induced localized apical F-actin accumulations in control and
treated Caco-2 cellsa
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Effect of LB-SCS on S. enterica serovar Typhimurium
SL1344-induced cytokine secretion in Caco-2/TC-7 cells.
It is
known that the association of serovar Typhimurium with epithelial cells
in vitro is followed by the induction of chemotactic-cytokine secretion
(8, 19, 22). We examined whether the LB-SCS was able to
modify Salmonella-induced IL-8 secretion (Fig.
5).
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FIG. 5.
Effect of LB-SCS treatment on S. enterica
serovar Typhimurium SL1344-induced IL-8 secretion in Caco-2/TC-7 cells.
Each value shown is the mean ± SEM of values from three
experiments (three successive passages of cultured cells). Statistical
analysis was performed with Student's t test. (A) Effect of
MRS-HCl, MRS-LA, and LB-SCS treatments on SL1344-induced IL-8 secretion
when the infecting bacteria were pretreated prior to infection. There
was a significant difference (P < 0.01) between
control SL1344 values and those for MRS-HCl or MRS-LA treatment. There
was also a highly significant difference (P < 0.001)
between control SL1344 values and those for LB-SCS. (B) Effect of
MRS-HCl, MRS-LA, and LB-SCS treatments on SL1344-induced IL-8 secretion
in preinfected cells. There was not a significant difference between
control SL1344 values and those for MRS-HCl, MRS-LA, or LB-SCS
treatment.
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We exposed serovar Typhimurium (5 × 10
7 CFU/ml) to
LB-SCS, MRS-HCl, or MRS-LA (1 h of treatment) prior to the cell
infection.
As shown in Fig.
5A, a marked decrease in SL1344-induced
IL-8
secretion was observed when the Caco-2/TC-7 cells were infected
with the LB-SCS-pretreated
S. enterica serovar Typhimurium.
Indeed,
we found a highly significant (95%) decrease in IL-8 secretion
compared with the control cell monolayer infected with untreated
salmonellae. It was noticed that MRS-LA and MRS-HCl had intrinsic
significant activity but that it was lower than that of LB-SCS.
We observed that when the Caco-2/TC-7 cells were preinfected with
SL1344 (5 × 10
7 CFU/well) and then treated with
LB-SCS for 2 h, no change in
the
Salmonella-induced
IL-8 secretion was observed compared with
the cell control infected
with the untreated pathogen (Fig.
5B).
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DISCUSSION |
As recently discussed (25), a major problem in
antibiotic therapy is the ineffectiveness of these drugs against
pathogens that have already entered a host cell. For example, several
antibiotics are not able to penetrate host cells and are only effective
against extracellular pathogens (26), whereas other
molecules are effective against intracellular bacteria (25).
For antibiotics capable of crossing the epithelial membrane, the
problem resides in the capacity of the compound to reach the particular
intracellular location in which the pathogen develops its intracellular
lifestyle, which includes surviving, proliferating, intracellular
movement, alteration of cell functions, and/or induction of cell apoptosis.
We have previously reported that the human L. acidophilus
strain LB develops antimicrobial activity against a wide range of gram-negative and gram-positive pathogens in vitro and in vivo (2,
4-6). To gain further insight into the mechanism by which L. acidophilus LB inhibits Salmonella infection
in vitro and in vivo (5), we have used polarized epithelial
cell monolayers creating impermeable epithelial barriers, i.e., the
cultured human adenocarcinoma cell line Caco-2/TC-7 (1),
which is a model of the mature enterocyte of the small intestine
(23). Attachment to the intestinal brush border (9,
16) prior to cell entry is a prerequisite for S. enterica serovar Typhimurium pathogenesis. Contact between the
pathogen and the host cell is followed by synthesis of bacterial
proteins, eliciting host cell signaling followed by profound
cytoskeletal rearrangements, bacterium internalization, and
intracellular bacterial proliferation (for a review, see reference 13). Observations in the present study complete our
previous reports (4, 5) and explain how the LB-SCS
antagonizes serovar Typhimurium infection in vitro and in vivo. Indeed,
we demonstrated that LB-SCS treatment kills the intracellular
salmonellae. Moreover, our results demonstrate that by killing the
intracellular pathogen, the secreted antimicrobial compound(s) present
in the LB-SCS promotes the decrease in serovar Typhimurium-induced cell lesions.
Many studies have demonstrated that the characteristic response of
epithelial cells of the intestinal mucosa to bacterial adhesion is the
release of a range of cytokines, such as IL-6, -7, -8, and -10 and
tumor necrosis factor alpha (for a review, see reference
27). For example, infection of the human embryonic intestinal cell line INT407 by S. enterica serovar
Typhimurium SL1344 induces host cell signal transduction involving a
cascade of the three mitogen-activated protein kinases ERK, JNK, and
p38 (19). Induction of these signaling pathways leads to the
activation of the transcription factors NF-
B and AP-1, resulting in
the production of proinflammatory cytokines such as IL-8. IL-8, a member of the C-X-C family of chemotactic cytokines, is a potent chemoattractant for polymorphonuclear leukocytes developing
microbicidal activity involving both oxidative and nonoxidative
pathways in host defense systems. We found here that infection of
polarized Caco-2/TC-7 cells by serovar Typhimurium SL1344 is followed
by the release of IL-8. We observed that LB-SCS treatment exerts the
opposite effect on Salmonella-induced IL-8 secretion as a function of the experimental conditions. Inhibition of IL-8 secretion was found when SL1344 was treated with LB-SCS prior to cell infection. We have previously reported that under these conditions of infection the LB-SCS treatment inhibits the association of serovar Typhimurium with Caco-2 cells (5). Our present results demonstrate that inhibition of bacterial adhesion could promote the blockage of the
adhesion-dependent, S. enterica serovar Typhimurium-induced IL-8 release (8, 19, 27). An opposite effect was observed when LB-SCS treatment was applied to SL1344-preinfected Caco-2/TC-7 cells, since no inhibition of serovar Typhimurium-induced IL-8 release
was found. Under these conditions, S. typhimurium
preliminarily interacted with and then penetrated the cell, inducing
IL-8 production prior to LB-SCS treatment. Altogether, our results
indicate that LB-SCS treatment did not interfere directly with IL-8
synthesis but could block adhesion-dependent cytokine production by
acting directly against the pathogen.
In conclusion, we have obtained consistent data to explain how
LB-SCS, containing a secreted antimicrobial compound(s)
(5), is able to inhibit the intracellular lifestyle of
S. enterica serovar Typhimurium infecting human polarized
intestinal cells.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Unité 510, Faculté de Pharmacie, Université Paris XI, F-92296
Châtenay-Malabry, France. Phone: (33) 1.46.83.56.61. Fax: (33)
1.46.83.56.61. E-mail: alain.servin{at}cep.u-psud.fr.
| |
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Applied and Environmental Microbiology, March 2000, p. 1152-1157, Vol. 66, No. 3
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Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
