RESEARCH ARTICLE
Mannose-speci¢c interaction of Lactobacillus plantarum with
porcine jejunal epithelium
Gabriele Gross1,2, Jan van der Meulen1, Johannes Snel2, Roelof van der Meer2, Michiel Kleerebezem2,3,
Theo A. Niewold1,4, Marcel M. Hulst1 & Mari A. Smits1
1
Animal Breeding and Genomics Centre, Animal Sciences Group of Wageningen UR, Lelystad, The Netherlands; 2Health and Safety, NIZO Food
Research, Ede, The Netherlands; 3Laboratory of Microbiology, Wageningen University, Dreijenplein, Wageningen, The Netherlands; and 4Nutrition and
Health, Katholieke Universiteit Leuven, Kasteelpark Arenberg, Heverlee, Belgium
Correspondence: Mari A. Smits, Animal
Breeding and Genomics Centre, Animal
Sciences Group of Wageningen UR, PO Box
65, 8200 AB Lelystad, The Netherlands. Tel.:
131 320 238270; fax: 131 320 238050;
e-mail: [email protected]
Received 27 February 2008; revised 9 June
2008; accepted 5 July 2008.
First published online 31 July 2008.
DOI:10.1111/j.1574-695X.2008.00466.x
Editor: Patrik Bavoil
Keywords
Lactobacillus plantarum ; host–microorganism
interaction; PAP/RegIII; msa/srtA ; gene
expression; probiotic.
Abstract
Host–microorganism interactions in the intestinal tract are complex, and little is
known about specific nonpathogenic microbial factors triggering host responses in
the gut. In this study, mannose-specific interactions of Lactobacillus plantarum
299v with jejunal epithelium were investigated using an in situ pig Small Intestinal
Segment Perfusion model. The effects of L. plantarum 299v wild-type strain were
compared with those of two corresponding mutant strains either lacking the gene
encoding for the mannose-specific adhesin (msa) or sortase (srtA; responsible for
anchoring of cell surface proteins like Msa to the cell wall). A slight enrichment of
the wild-type strain associated with the intestinal surface could be observed after
8 h of perfusion when a mixture of wild-type and msa-mutant strain had been
applied. In contrast to the mutant strains, the L. plantarum wild-type strain tended
to induce a decrease in jejunal net fluid absorption compared with control
conditions. Furthermore, after 8 h of perfusion expression of the host gene
encoding pancreatitis-associated protein, a protein with proposed bactericidal
properties, was found to be upregulated by the wild-type strain only. These
observations suggest a role of Msa in the induction of host responses in the pig
intestine.
Introduction
The intestinal microbial community contains a complex
system of bacterial species, maintaining a variety of interactions with the host via the intestinal epithelium. For
example, commensal microorganisms have been recognized
to influence the intestinal immune system and to induce the
expression of bactericidal proteins that can shape the
intestinal microbial community (Hooper et al., 2003; Macpherson & Harris, 2004; Cash & Hooper, 2005). Probiotic
lactic acid bacteria are intended to have a beneficial
health effect for the host and to improve resistance against
intestinal pathogens (Cross, 2002; Reid & Burton, 2002).
Currently, transcriptome profiles are increasingly used to
gain global views on host responses to bacterial stimuli reflected
in gene expression levels. Colonization of germ-free mice with
commensal and probiotic bacteria such as Bacteroides thetaiotaomicron and Bifidobacterium longum has been shown to cause
FEMS Immunol Med Microbiol 54 (2008) 215–223
substantial changes in gene expression of the host, including the
induction of host genes involved in innate immunity, mucosal
barrier fortification, and intestinal maturation (Hooper et al.,
2001; Sonnenburg et al., 2006).
Intestinal carbohydrate moieties have been demonstrated to
play an important role in the interaction between numerous
bacterial species and their hosts (Neeser et al., 2000; Karlsson,
2001). For example, it has been demonstrated for certain
Lactobacillus plantarum strains and type 1-fimbriated pathogens such as Salmonella enterica and enterotoxigenic Escherichia
coli that they have the capacity to bind to mannose-containing
sugar moieties on the surface of epithelial cells (Wold et al.,
1988; Aslanzadeh & Paulissen, 1992; Adlerberth et al., 1996).
Lactobacillus plantarum is a member of the human intestinal
microbiota and specific strains are marketed as probiotics in
functional foods with a proposed beneficial health effect (for a
review, see De Vries et al., 2006). Furthermore, the mannosespecific adhesin-encoding gene of L. plantarum, msa, has been
2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
216
identified recently (Pretzer et al., 2005). This gene is of interest
because it represents a potential probiotic function of
L. plantarum that might be relevant for increased resistance
against intestinal infections by competitive exclusion of pathogens (Adlerberth et al., 1996; Lee & Puong, 2002).
The aim of this study was to gain first insights into the role
of mannose-specific interactions of L. plantarum for its capacity to trigger responses of the host epithelial cells. Therefore,
the effects of L. plantarum 299v wild-type strain in the intestine
were compared with those of two gene-specific mutant strains,
lacking either the functional msa or the srtA (encoding sortase)
gene. Because Msa belongs to the sortase target proteins of
L. plantarum, it cannot be anchored properly to the cell wall in
an srtA-deficient strain that concomitantly has lost its mannose-adhesion capacity, analogous to the msa mutant (Pretzer
et al., 2005). Bacterial association with the epithelial surface,
intestinal net fluid absorption, and the induction of differential
gene expression of the host by the wild-type and mutant strains
were studied in a pig in situ jejunal loop model, the Small
Intestinal Segment Perfusion model (SISP). This model was
chosen because of the similarities between pig and human
intestinal physiology and anatomy and the possibility to study
the very early intestinal response to various food components
in situ in a single animal providing an isogenic background
under nearly physiological conditions (Bruins et al., 2006; Kiers
et al., 2006). Samples were taken after 4 and 8 h of perfusion of
jejunal loops with bacterial suspensions, representing an early
response to the experimental treatments. The results of this
study provide first important insights into the molecular
interaction mechanisms of probiotics with the host.
Materials and methods
Animals
Four weaned male 5-week-old pigs (Duroc Topigs 20)
were obtained from a commercial Dutch piggery (Varkens-
G. Gross et al.
bedrijf Bert Rijnen, Oirschot, The Netherlands). After transport to the experimental facilities, animals were continued to
be fed ad libitum with standard liquid pig feed, prepared at
the piggery, and were pretreated with colistine (Eurovet
Animal Health, Bladel, The Netherlands) for 4 days according to the manufacturers’ recommendations to standardize
background microbiota. Six days after arrival, animals were
fasted overnight before the SISP experiment was performed.
The animal study was approved by the local Animal Ethics
Commission in Lelystad, The Netherlands, in accordance
with the Dutch Law on Animal Experimentation.
SISP test
The SISP test was performed essentially as described by
Nabuurs et al. (1993) and Niewold et al. (2005). Briefly, pigs
were sedated with azaperone and inhalation anesthesia was
applied with a gas mixture of oxygen, nitrous oxide, and
isoflurane. The abdominal cavity was opened and five pairs
of segments, each 20 cm long and with inlet tubes at the
cranial side and outlet tubes at the caudal side of each
segment, were prepared at 30–50% of the jejunum. Before
further treatment, samples were taken from the jejunum
cranially of segment 1, between segments 5 and 6, and
caudally of segment 10.
The experiment was started by injecting 20 mL of
L. plantarum suspensions [wild-type, msa, or srtA mutant;
1010 CFU mL1 in phosphate-buffered saline (PBS)] or PBS
without bacteria as a control into the segments, respectively.
Salmonella typhimurium (5 mL suspension, 109 CFU mL1
in PBS) was used as a positive control for gene expression
analysis, under the same conditions as used in an earlier
experiment (Niewold et al., 2007). Figure 1 gives a schematic
overview of the treatment and samplings per segment. In the
four animals, the order of positions was shuffled per time
point to avoid local effects, except for segments treated with
Fig. 1. Schematic overview of treatments per jejunal segment as applied in the SISP test. PBS, control; Lp wt, Lactobacillus plantarum 299v wild-type
strain; Lp msa ko, L. plantarum 299v msa-mutant strain; Lp srtA ko, L. plantarum 299v srtA-mutant strain; St, Salmonella typhimurium DT104 (only
sampled at time point 8 h as a positive control for gene expression analysis); Lp wt1msa ko, L. plantarum 299v wild-type strain 50%1msa-mutant strain
50% (only sampled at time point 8 h for microbiological analysis). Because pig 3 died unexpectedly during the SISP procedure, sampling after 8 h was
not possible for this animal. Segements treated with Salmonella and the mixture of L. plantarum 299v wild type and msa mutant were sampled after 4 h,
in contrast to the other three animals.
2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
FEMS Immunol Med Microbiol 54 (2008) 215–223
217
Interaction of L. plantarum with pig intestinal epithelium
S. typhimurium or with a 50–50% mixture of L. plantarum
wild-type and msa-mutant strain. These treatments were
included as a positive control for gene expression and for
microbiological analysis only, respectively. Segments
sampled at one time point were arranged in local proximity
due to differences in gene expression along the intestine.
After 1 h, the segments were perfused with perfusion fluid
(physiological salt solution with 0.1% glucose and 0.1%
amino acids), 2 mL injected every 15 min for 3 or 7 h.
Effluent fluid was collected during this time. After a total of
4 h, some segments were removed and samples were taken
(see Fig. 1), and after a total of 8 h the remaining segments
were sampled and pigs were euthanized. Fluid remaining in
each segment was added to the effluent. The length and
diameter of each segment was measured for calculation of
net fluid absorption in mL cm2 [difference between in- and
outflow divided by the surface area (length circumference) of a segment]. Mucosal scrapings from the segments
were collected, immediately frozen in liquid nitrogen, and
stored at 70 1C until further analysis. As described below,
RNA was extracted from these scrapings to specifically assess
gene expression in epithelial cells, which are supposed to be
in direct contact with luminal bacteria. Furthermore, 1 cm2
pieces were cut from the intestinal wall with a stamp, washed
in PBS, and stored at 4 1C in PBS until they were
homogenized and plated in serial dilutions on appropriate
agar plates.
Bacterial strains and culture media
Lactobacillus plantarum 299v was initially used as the wildtype strain (Johansson et al., 1993). Isogenic msa- and srtAmutant derivatives of this strain had been constructed
previously (G. Gross, J. Snel, J. Boekhorst, M.A. Smits, &
M. Kleerebezem, unpublished data). Briefly, msa and srtA
deletions had been accomplished by stable double-crossover
cat replacement of the target genes in the chromosome of
L. plantarum 299v wild type similar to previous construction of msa and srtA mutants in strain WCFS1 (Pretzer et al.,
2005). Both wild-type strains, 299v and WCFS1, are capable
of mannose adhesion as determined in a yeast agglutination
assay, whereas deletion of msa or srtA leads to a complete
loss of agglutination ability in each of the mutant strains
(Pretzer et al., 2005; G. Gross, J. Snel, J. Boekhorst, M.A.
Smits & M. Kleerebezem, unpublished data). Before the
SISP experiment, the L. plantarum 299v wild-type, msa–
mutant, and srtA-mutant strains were selected for natural
rifampicine resistance by culturing on medium with increasing concentrations of that antibiotic (Bron et al., 2004).
Salmonella typhimurium DT104A (Hendriksen et al., 2004)
served as a positive control in the SISP test because the
effects of this strain on host gene expression have already
been determined using this model (Niewold et al., 2007).
FEMS Immunol Med Microbiol 54 (2008) 215–223
For use in the SISP model, L. plantarum strains were
grown overnight at 37 1C in Man Rogosa Sharp (MRS)
broth (Merck, Darmstadt, Germany) containing 50 mg mL1
rifampicine for the wild-type strain and additionally
7 mg mL1 chloramphenicol for the msa- and srtA-mutant
strains. Salmonella typhimurium was grown overnight
aerobically at 37 1C in Luria–Bertani broth (BD Difco,
Alphen aan den Rijn, The Netherlands). Bacterial counts
of the initial suspensions used in the experiment were
standardized by OD measurement and determined by plating
of serial dilutions on appropriate agar plates. A mixture of
L. plantarum 299v 50% wild-type strain and 50% msa-mutant
strain, included for additional microbiological analyses after
8 h of perfusion, was prepared by adding the same volumes of
adjusted suspensions of both strains to each other.
Serial dilutions of homogenized jejunal samples
(1 cm2 pieces) from the SISP test were plated on MRS agar
containing 50 mg mL1 rifampicine that were incubated
anaerobically at 37 1C for 48 h before CFU of lactobacilli
were counted. One hundred CFU cultured from segments
treated with the mixture of L. plantarum 299v wild-type and
msa-mutant strain were replica plated on MRS agar containing 50 mg mL1 rifampicine and 7 mg mL1 chloramphenicol.
Thereby, the two strains could be distinguished, i.e. the wild
type being resistant only to rifampicine and the msa mutant
being resistant to both rifampicine and chloramphenicol.
Using this method, it was also confirmed that the initial
bacterial suspension contained equal proportions of each of
the two strains.
Isolation of total RNA and microarray analysis
Total RNA was isolated from c. 0.5–1 g of frozen mucosal
scrapings of the jejunal segments from the SISP test. The
tissue was homogenized in 4 mL TRIzol reagent (Invitrogen,
Breda, the Netherlands) and the extractions were essentially
performed according to the instructions of the manufacturer with additional steps to remove proteoglycan and
polysaccharide contaminations and DNAse treatment as
described earlier (Niewold et al., 2005). Finally, RNA was
dissolved in RNAse-free water and stored at 70 1C until
further use. All samples were checked by agarose gel
electrophoresis and UV spectrophotometry. For microarray
analysis, RNA pools were prepared by mixing equal amounts
of RNA isolated from intestinal segments of the different
pigs collected at the same time point with the same bacterial
treatment.
For gene expression analysis, a home-made porcine
cDNA microarray was used containing 3072 clones from a
jejunum-expressed sequence tag (EST) library (Niewold
et al., 2005) plus 3072 clones from a spleen EST library, all
spotted in quadruplicate. For production of the spleen ESTs,
spleens were collected from the same four 12-week-old pigs
2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
218
that provided jejunal mucosal scrapings for preparation of
the jejunum array (Niewold et al., 2005). A total of 2688
EST clones generated from total RNA isolated from pooled
spleen tissue (n = 4) were spotted on the array along with
192 cDNA clones selected from Marc 1 and Marc 2
libraries (Fahrenkrug et al., 2002) and 192 EST clones
prepared from RNA isolated from in vitro ConA/lipopolysaccharide-stimulated spleen cells derived from the abovedescribed pooled spleen tissue. Dual-color hybridization
of the slides was performed using the RNA MICROMAX
TSA labeling and detection kit (Perkin-Elmer, Zoetermeer,
The Netherlands) as described earlier (Niewold et al.,
2005). Briefly, labeled cDNA was prepared from 1 mg of
RNA template from the pools of the control and of the
bacterial treatments of one time point, respectively, using
biotin and fluorescein. Labeled cDNAs were simultaneously hybridized to a microarray slide and detected with
Cy5 (biotin) and Cy3 (fluorescein), respectively. For each
comparison, a dye-swap was performed as a second
hybridization with reversed labeling. The slides were
scanned for Cy5 and Cy3 fluorescence in a Packard
Bioscience BioChip Technologies apparatus (ScanArray
Express, Perkin-Elmer). The image was processed with
SCANARRAY EXPRESS software (Perkin-Elmer), automatically
gridding the spots and measuring spot intensities. Manual
elimination of irregular-shaped spots was performed in
GenePix Pro 5.0 (Molecular Devices, Apeldoorn, The
Netherlands). Data were normalized (Blank-specific background correction; Lowess fit function with a fraction of
0.2) using a customized version of the statistical software
package R (Yang et al., 2002; B. Hulsegge, M.F.W. te Pas &
M.H. Pool, presented at the 2nd Netherlands Bioinformatics Conference/4th International Symposium on Networks in Bioinformatics, Amsterdam, The Netherlands,
16–19 April 2007). Dye-swaps were simultaneously analyzed using significance analysis of microarrays (SAM;
Tusher et al., 2001). Additionally, significantly differential
expressed spots with an M value of o 1.58 or 4 1.58
[M = log2 (Cy5/Cy3)] were selected manually from data
reports generated after normalization, i.e. with an absolute
ratio of the effects of a bacterial treatment vs. control of
greater than threefold. The use of the home-made porcine
microarray technically implies a threshold level of
4 3 -/ o 0.33 -fold changes of differential gene expression to attain reliable data. For each probe present on
the array eight values could be obtained, four spots per
slide and per dye-swap. Probes with more than three
missing values out of eight were removed from further
analysis.
The data discussed in this publication have been deposited
in NCBIs Gene Expression Omnibus (GEO, http://www.
ncbi.nlm.nih.gov/geo/) and are accessible through GEO Series
accession number GSE9209.
2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
G. Gross et al.
Reverse transcriptase reaction and quantitative
real-time PCR
cDNA was reverse transcribed from all individual and
pooled RNA samples using Superscript II reverse transcriptase (Invitrogen) according to the manufacturer’s
recommendations. Pancreatitis-associated protein (PAP)
expression levels were quantified in all samples using the
LightCycler Real-Time PCR (Roche Diagnostics, Almere,
The Netherlands) using SybrGreen as described earlier
(Niewold et al., 2007). The concentration of the 18S rRNA
gene was determined accordingly by RT-PCR and was used
to normalize the relative amount of PAP mRNA in all
samples.
Statistical analysis
When appropriate, results are indicated as SE of the mean
(SEM) and the significance of the difference between the
results of the treatment and the control conditions was
calculated using Student’s t-test (two sided; considered
statistically significant when P is 0.05; trends are indicated when P is 0.1). Changes in the ratio of wild type
and msa-mutant strain CFU when applied together in a
50–50% mixture were assessed per animal using the sign test
(two sided; a = 0.05; considered statistically significant when
P is 0.05).
Results
Although all animals were healthy at the start of the
experiment, animal three died unexpectedly during the SISP
procedure after sampling at 4 h. From this pig, segments in
which Salmonella and the mixture of L. plantarum 299v
wild type and msa mutant had been applied were also
sampled additionally at 4 h in contrast to the other three
animals. However, further inspection revealed no abnormalities of this pig and, therefore, it was not excluded from
data analysis of time point 4 h (i.e. 4 h n = 4, 8 h n = 3).
Microbiological analyses
Bacterial counts of L. plantarum associated with the intestinal surface were determined by serial plating of jejunal
samples, cut from the intestinal wall, on MRS agar containing 50 mg mL1 rifampicine (Fig. 2). In the control segments,
background values of apparently naturally rifampicineresistant lactobacilli were detected. Numbers of CFU were
statistically significantly higher in all jejunal segments in
which L. plantarum was applied compared with the control
segments after 4 and 8 h of perfusion (P o 0.05 for all
treatments vs. control). No difference in bacterial counts
could be observed between the treatments with L. plantarum
299v wild type, both msa- and srtA-deletion strains, and
with the mixture of wild-type and msa-deletion strain.
FEMS Immunol Med Microbiol 54 (2008) 215–223
219
Interaction of L. plantarum with pig intestinal epithelium
1000
net fluid absorption
(µL cm–2)
800
#
600
200
*
8.0
*
* *
*
*
7.0
*
6.0
sa
St
ko
ko
tA
t+
m
Lp
w
sa
m
sr
Lp
t
w
Lp
Fig. 4. Net fluid absorption per segment expressed in mL cm2 SEM.
Data after 4 h of perfusion are depicted with white bars (n = 4), and data
after 8 h of perfusion are depicted with gray bars (n = 3). Trends towards
statistically significant differences between treatments compared with
control conditions are indicated with a number sign (time point 8 h: PBS
(control) vs. Lactobacillus plantarum wild type P = 0.087, PBS (control) vs.
Salmonella typhimurium P = 0.070).
5.0
ko
ko
w
Lp
t+
sr
m
sa
tA
ko
sa
m
Lp
PB
S
Lp
(c
on
Lp
tro
w
l)
t
4.0
Fig. 2. Numbers of lactobacilli determined by serial plating of jejunual
samples on MRS agar containing 50 mg mL1 rifampicine. Results are
calculated as log10 CFU cm2 jejunal segment SEM. Data after 4 h of
perfusion are depicted with white bars (n = 4), and data after 8 h of
perfusion are depicted with gray bars (n = 3). Significantly different
results (P o 0.05) compared with the control segments sampled at the
same time point are indicated with an asterisk.
for all segments, reflecting a direct flow of fluid through the
segments in the beginning of the experiment caused by the
initial injection of the suspensions. After 8 h of perfusion, a
trend towards a statistically significant decrease in net fluid
absorption compared with the control conditions could be
observed in the segments treated with L. plantarum wildtype strain and S. typhimurium (P = 0.087 and P = 0.070,
respectively). Absorption after treatment with both msaand srtA-mutant strains and with the 50–50% mixture of
wild-type and msa-mutant strain was at the same level as
that of the control segments.
Gene expression analysis
70
*
*
60
% CFU
Lp
on
tro
log10 CFU cm−2
jejunal segment
9.0
PB
S
Calculated net fluid absorption per segment is illustrated in
Fig. 4. The results after 4 h of perfusion are almost identical
ko
0
(c
Net fluid absorption
#
400
l)
However, additional information can be gathered in a
competitive setting between bacterial strains that share the
same binding sites for attachment to intestinal cells (Larsen
et al., 2007). Replica plating discriminating between the two
strains in the mixture revealed a modest enrichment of the
wild-type strain in three out of four animals compared with
the initial bacterial suspension (Fig. 3; statistically significant for animals 3 and 4, P 0.05).
50
40
30
4
An
im
al
3
An
im
al
2
An
im
al
1
al
im
An
In
iti
al
su
sp
.
20
Fig. 3. Percentage of CFU from replica plating of jejunal segments
treated with a mixture of Lactobacillus plantarum 299v 50% wild-type
and 50% msa-mutant strain, per individual animal. Wild-type L. plantarum is depicted with white bars, and msa mutant with gray bars. Animal 3
was sampled after 4 h of perfusion, and the other three animals after 8 h.
Statistically significant changes in the ratio of wild-type and mutant
strain as compared with the initial suspension (indicated by ‘Initial susp.’)
that had been injected into the segments are indicated with an asterisk
(P 0.05).
FEMS Immunol Med Microbiol 54 (2008) 215–223
For microarray analysis, pooled samples of segments with the
same bacterial treatments were compared with control segments sampled at the same time point, and a threshold level of
4 3 -/ o 0.33 -fold changes of differential gene expression
was applied for all comparisons because a lower cut-off value is
technically not appropriate for the home-made microarray
used in this study. Detected differences in host gene expression
are presented in Table 1. No greater than threefold differences
in host gene expression were revealed between control and any
of the L. plantarum strains at time point 4 h. However, after 8 h
of perfusion only the expression of one gene – encoding PAP –
was detected to be greater than threefold (10.6 ) upregulated
by L. plantarum 299v wild type. In contrast, treatment with
L. plantarum 299v msa- and srtA-mutant strains did not cause
any greater than threefold differential gene expression after 8 h
perfusion as compared with the control treatment. No downregulation of gene expression (ratio o 0.33) was observed for
any bacterial treatment.
2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
220
Table 1. Differential gene expression induced after 8 h of perfusion of
jejunal segments with bacterial suspensions
Comparison
L. plantarum 299v wild type vs.
control
L. plantarum 299v msa mutant vs.
control
L. plantarum 299v srt mutant vs.
control
S. typhimurium vs. control
Samples taken at the beginning of the
experiment (t = 0) vs. control
Differentially expressed
genes at time point 8 h
Pancreatitis-associated
protein (PAP; 10.6 )
No differential gene
expression
No differential gene
expression
Pancreatitis-associated
protein (PAP; 11.2 )
Matrix-metalloproteinase-1
(MMP-1; 3.4 )
Chemokine CXC-like protein
(IP-10; 4.1 )
Interleukin 8 (IL-8; 2.0 )
b-Globin (0.2 )
Cytochrome P450
(CYP3A29) (0.3 )
Calbindin (0.4 )
No up- or downregulation of gene expression was found after 4 h of
perfusion. Results from microarray analysis of pooled samples from
experimental treatment segments vs. control segments at the same time
point are indicated as differentially expressed genes (ratio 4 3 /
o 0.33 ; -fold change).
Pools from samples of segments perfused with Salmonella typhimurium
and from samples collected at the beginning of the experiment were
only compared vs. control segments at time point 8 h, the latter serving
as a control for the effect of the perfusion procedure itself.
The results of microarray comparisons of gene expression
in segments perfused with S. typhimurium for 8 h vs. control
segments are displayed in Table 1 and are in agreement with
previous findings (Niewold et al., 2007). Data analysis using
SAM indicated IL-8 mRNA and calbindin mRNA to be
differentially expressed although these did not fulfill the
selection criterion of greater than threefold differential
regulation. An explanation for this is that SAM processes
data in a different way compared with the manual selection
from normalized data, e.g. regarding averaging of M-values
and exclusion of spots. Slight changes in gene expression
caused by the perfusion process itself, assessed by comparison of pooled samples taken at the beginning of the
experiment vs. control segments at time point 8 h, were also
similar to earlier results using the SISP model (Niewold
et al., 2007).
Results obtained by microarray analysis indicating an
induction of PAP mRNA expression by the L. plantarum
299v wild-type strain, but not by the msa- and srtA-mutant
strains after 8 h of perfusion were confirmed using quantitative RT-PCR (Fig. 5). Analysis of individual samples of the
animals demonstrated that PAP mRNA expression displayed
a large interindividual variation among the animals;
2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
PAP expression normalized for 18S
(arbitrary units)
G. Gross et al.
25
20
15
10
5
0
124P
124P
124P
124P
124P
PBS (control)
Lp wt
Lp msa ko
Lp srt ko
St
Fig. 5. Relative expression of PAP mRNA after 8 h of perfusion in pools
and per individual sample as assessed by quantitative RT-PCR normalized
for the amount of 18S rRNA gene. Individual animals are depicted with
white bars (animals 1, 2, and 4 indicated with 1, 2, 4 beneath bars) and
pooled samples with gray bars (indicated with P beneath bars).
especially, expression levels in animal 2 were low under all
experimental conditions.
Discussion
In this study, a bacterial wild type and two gene-specific
mutant strains of L. plantarum were used to study specific
host–microorganism interaction mechanisms. The role of
mannose-specific host–microorganism interactions was
explored, including the effect of these bacterial strains on
host responses such as fluid absorption and host gene
expression. The findings of this study indicate that in this
model, L. plantarum can interact with the host by inducing
mRNA expression of the innate immune factor PAP and
compromise fluid absorption of the intestine in a manner
resembling that of the pathogen S. typhimurium. Importantly, the results imply that the mannose-specific adhesin of
L. plantarum mediates these host–microorganism interactions. As various bacterial species are capable of mannosespecific interactions, the observations presented here might
be of interest for several other probiotic, commensal, or
pathogenic microorganisms. Intriguingly, the proposed
mannose-specific bacterial induction of PAP mRNA expression suggests a newly identified molecular mechanism of
host–microorganism communication.
Previously, it had been concluded from an in vitro yeast
agglutination assay that the L. plantarum gene msa is
directly involved in mannose adhesion (Pretzer et al.,
2005). In contrast to L. plantarum 299v wild-type, genespecific msa- or srtA-mutant strains are not capable of
mannose-binding in vitro (G. Gross, J. Snel, J. Boekhorst,
M.A. Smits & M. Kleerebezem, unpublished data). In the
current study, a competitive advantage of L. plantarum 299v
wild type compared with the mutant strain lacking msa was
demonstrated in jejunal segments perfused with a 50–50%
mixture of both strains. This enrichment of the wild-type
FEMS Immunol Med Microbiol 54 (2008) 215–223
221
Interaction of L. plantarum with pig intestinal epithelium
strain in a competitive setting with the msa mutant is the
first indication of a contribution of Msa to the attachment of
L. plantarum to the host-intestinal surface in situ. The
enrichment is not likely to be caused by growth differences
between the wild-type and the mutant strain because a
corresponding effect was not observed after up to 24 h of in
vitro coculturing of both strains (data not shown). It should
be noted that adhesion of lactobacilli to the intestinal
surface is a result of multifactorial interactions, involving
e.g. surface-layer proteins, lipoteichoic acids, and aggregation ability (Granato et al., 1999; Kos et al., 2003; JohnsonHenry et al., 2007). Therefore, mechanisms other than
mannose-specific ones could have facilitated bacterial
adherence in this study. As demonstrated here, a competitive
setting is appropriate to detect differences between bacterial
strains that recognize the same binding sites for attachment
to the intestinal epithelium because these slight differences
could not be detected in segments perfused with the
individual bacterial strains.
Net fluid absorption in segments perfused with L. plantarum 299v wild-type strain and S. typhimurium was found
to be decreased as compared with the control segments and
those treated with the L. plantarum-mutant strains. Reduced
net fluid absorption can be caused by inhibition of electrolyte absorption or by the physiological situation of secretory
diarrhea. These findings are known to be caused by a
pathogen like Salmonella, but are not anticipated for probiotic microorganisms like lactobacilli. Metabolic activity of
bacteria and subsequent accumulation of organic acids such
as lactic acid have also been reported to induce net fluid
excretion (Saunders & Sillery, 1982; Argenzio & Meuten,
1991). However, this is not likely to be an explanation for
the effect observed in the present study because a decrease of
fluid absorption was only caused by L. plantarum wild type,
but not by equal numbers of the two mutant strains,
suggesting a particular Msa-mediated host response. It can
be hypothesized that Msa triggers decreased net fluid
absorption as an initial innate reaction of the host against
increased bacterial penetration. Possibly, the exposure to the
lower bacterial load with Msa-expressing lactobacilli in the
injected bacterial suspension can explain why the mixture of
wild-type and msa-mutant strain did not affect net fluid
absorption.
In this model, an increased expression of host PAP mRNA
was observed in two of the three individual animals after
treatment with the L. plantarum 299v wild-type strain.
Intriguingly, in the same animals the msa- and srtA-mutant
strains did not induce a corresponding upregulation of PAP
mRNA expression. This remarkable finding suggests that
PAP mRNA induction is related to the functional characteristics of Msa. PAP/RegIII is a C-type lectin that has originally
been described to be induced in acute pancreatitis and
inflammatory bowel disease (Iovanna et al., 1993; Unno
FEMS Immunol Med Microbiol 54 (2008) 215–223
et al., 1993; Dieckgraefe et al., 2002). PAP/RegIII plays a role
in the promotion of epithelial cell proliferation, growth and
regeneration of intestinal tissue, and in the inflammatory
response (Moucadel et al., 2001; Vasseur et al., 2004;
Breikers et al., 2006). Recently, it has been shown that PAP/
RegIII binds to peptidoglycans on the surface of grampositive bacteria and is directly antimicrobial (Cash et al.,
2006). Upregulation of PAP mRNA by L. plantarum wild
type as observed in the present study corresponds with the
assumption that PAP/RegIII is induced by increased bacterial–epithelial contact to limit potential microbial penetration and to maintain mucosal integrity. This is augmented
by its antimicrobial action and its contribution to epithelial
repair (Keilbaugh et al., 2005; Cash et al., 2006). Indeed,
elevated expression of PAP mRNA is also part of the reaction
to different pathogens such as enterotoxigenic E. coli and
Salmonella species as demonstrated in different animal
models (this study; Niewold et al., 2005, 2007; Rodenburg
et al., 2007). Furthermore, an increase in PAP/RegIII expression was detected in germ-free mice after colonization with
B. thetaiotaomicron, segmented filamentous bacteria, and
Schaedler’s E. coli, whereas Listeria innocua had no effect on
PAP/RegIIIg mRNA levels, and it was downregulated by
B. longum (Keilbaugh et al., 2005; Cash et al., 2006; Sonnenburg et al., 2006).
Remarkably, in pig 2 no enrichment of the wild-type
L. plantarum strain was observed after application of the
mixture of a wild-type and an msa-mutant strain (Fig. 3),
and hardly any PAP mRNA induction could be detected,
even after treatment with S. typhimurium (Fig. 5). Individual variation of intestinal PAP expression in pigs has been
observed under various experimental conditions (M. Hulst,
pers. commun.) and might be influenced by the overall
bacterial colonization level of each animal. This suggests
that the responsiveness on this parameter differs substantially per animal and that future studies will have to take into
account a proportion of ‘non-/late-responders’.
In the present study, very early host responses in the pig
intestine were analyzed upon challenge with L. plantarum.
Most other studies in this area focus on long-term changes
in gene expression induced by commensal or probiotic
microorganisms in humans and mice (Hooper et al., 2001;
Di Caro et al., 2005; Sonnenburg et al., 2006; Troost et al.,
2006). Therefore, in these studies substantially higher numbers of genes involved in various biological functions were
detected to be differentially expressed. In contrast to the
current study, these effects could not be linked to one
specific bacterial characteristic. Here, microarray analysis
revealed no downregulation of gene expression by the
bacterial treatments, and only one gene was found to be
induced by L. plantarum early after challenge. This is in
line with the expectation that the effects of commensal
or probiotic bacterial strains on gut response are more
2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
222
subtle compared with those induced during infections
with pathogens such as Salmonella that stimulate inflammatory pathways. Gene expression data after perfusion with
S. typhimurium as presented here are in agreement with this
and correspond well with earlier findings of Salmonellainduced host responses using the SISP model and the same
microarray platform (Niewold et al., 2007). Lower numbers
of genes found to be differentially expressed as compared
with other studies might be explained by a variety of
parameters, e.g. model host organism, differently chosen
threshold values for differential gene expression, differing
host cell/microorganism ratios, and technical aspects of the
home-made array platform in comparison with only
recently available commercial oligonucleotide arrays for pigs.
In conclusion, the results of the current study suggest a
role of Msa in bacterial adherence and induction of host
responses in the intestine, as reflected in decreased fluid
absorption and enhanced expression of PAP mRNA as an
innate immune parameter. Possibly, Msa-mediated host
responses might not necessarily be dependent on bacterial
adhesion properties. The preliminary indications that
mannose-specific interactions contribute to early host–
microorganism relationships and the proposed role of Msa
will be evaluated in further in vivo experiments using larger
numbers of animals.
Acknowledgements
The authors thank Johan Meijer and Arie Hoogendoorn for
assistance with the animal experiment and Agnes de Wit
for advice on performing microarray analyses and RT-PCRs.
This work was supported by the Centre for Human
Nutrigenomics, The Netherlands, and EADGENE (EU
contract no.: FOOD-CT-2004-506416).
References
Adlerberth I, Ahrné S, Johansson ML, Molin G, Hanson LÅ &
Wold AE (1996) A mannose-specific adherence mechanism in
Lactobacillus plantarum conferring binding to the human
colonic cell line HT-29. Appl Environ Microbiol 62: 2244–2251.
Argenzio RA & Meuten DJ (1991) Short-chain fatty acids induce
reversible injury of porcine colon. Dig Dis Sci 36: 1459–1468.
Aslanzadeh J & Paulissen LJ (1992) Role of type 1 and type 3
fimbriae on the adherence and pathogenesis of Salmonella
enteritidis in mice. Microbiol Immunol 36: 351–359.
Breikers G, van Breda SG, Bouwman FG, van Herwijnen MH,
Renes J, Mariman EC, Kleinjans JC & van Delft JH (2006)
Potential protein markers for nutritional health effects on
colorectal cancer in the mouse as revealed by proteomics
analysis. Proteomics 6: 2844–2852.
Bron PA, Grangette C, Mercenier A, de Vos WM & Kleerebezem
M (2004) Identification of Lactobacillus plantarum genes that
2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
G. Gross et al.
are induced in the gastrointestinal tract of mice. J Bacteriol
186: 5721–5729.
Bruins MJ, Cermak R, Kiers JL, van der Meulen J, van Amelsvoort
JM & van Klinken BJ (2006) In vivo and in vitro effects of tea
extracts on enterotoxigenic Escherichia coli-induced intestinal
fluid loss in animal models. J Pediatr Gastroenterol Nutr 43:
459–469.
Cash HL & Hooper LV (2005) Commensal bacteria shape
intestinal immune system development. ASM News 71: 77–83.
Cash HL, Whitham CV, Behrendt CL & Hooper LV (2006)
Symbiotic bacteria direct expression of an intestinal
bactericidal lectin. Science 313: 1126–1130.
Cross ML (2002) Microbes versus microbes: immune signals
generated by probiotic lactobacilli and their role in protection
against microbial pathogens. FEMS Immunol Med Microbiol
34: 245–253.
De Vries MC, Vaughan EE, Kleerebezem M & de Vos WM (2006)
Lactobacillus plantarum-survival, functional and potential
probiotic properties in the human intestinal tract. Int Dairy J
16: 1018–1028.
Di Caro S, Tao H, Grillo A, Elia C, Gasbarrini G, Sepulveda AR &
Gasbarrini A (2005) Effects of Lactobacillus GG on genes
expression pattern in small bowel mucosa. Dig Liver Dis 37:
320–329.
Dieckgraefe BK, Crimmins DL, Landt V, Houchen C, Anant S,
Porche-Sorbet R & Ladenson JH (2002) Expression of the
regenerating gene family in inflammatory bowel disease
mucosa: Reg Ia upregulation, processing, and antiapoptotic
activity. J Investig Med 50: 421–434.
Fahrenkrug SC, Smith TP, Freking BA et al. (2002) Porcine gene
discovery by normalized cDNA-library sequencing and EST
cluster assembly. Mamm Genome 13: 475–478.
Granato D, Perotti F, Masserey I, Rouvet M, Golliard M, Servin A
& Brassart D (1999) Cell surface-associated lipoteichoic acid
acts as an adhesion factor for attachment of Lactobacillus
johnsonii La1 to human enterocyte-like Caco-2 cells. Appl
Environ Microbiol 65: 1071–1077.
Hendriksen SW, Orsel K, Wagenaar JA, Miko A & van Duijkeren
E (2004) Animal-to-human transmission of Salmonella
Typhimurium DT104A variant. Emerg Infect Dis 10:
2225–2227.
Hooper LV, Wong MH, Thelin A, Hansson L, Falk PG & Gordon
JI (2001) Molecular analysis of commensal host-microbial
relationships in the intestine. Science 291: 881–884.
Hooper LV, Stappenbeck TS, Hong CV & Gordon JI (2003)
Angiogenins: a new class of microbicidal proteins involved in
innate immunity. Nat Immunol 4: 269–273.
Iovanna JL, Keim V, Bosshard A, Orelle B, Frigerio JM, Dusetti N
& Dagorn JC (1993) PAP, a pancreatic secretory protein
induced during acute pancreatitis, is expressed in rat intestine.
Am J Physiol 265: G611–G618.
Johansson ML, Molin G, Jeppsson B, Nobaek S, Ahrné S &
Bengmark S (1993) Administration of different Lactobacillus
strains in fermented oatmeal soup: in vivo colonization of
FEMS Immunol Med Microbiol 54 (2008) 215–223
223
Interaction of L. plantarum with pig intestinal epithelium
human intestinal mucosa and effect on the indigenous flora.
Appl Environ Microbiol 59: 15–20.
Johnson-Henry KC, Hagen KE, Gordonpour M, Tompkins TA &
Sherman PM (2007) Surface-layer protein extracts from
Lactobacillus helveticus inhibit enterohaemorrhagic Escherichia
coli O157: H7 adhesion to epithelial cells. Cell Microbiol 9:
356–367.
Karlsson KA (2001) Pathogen-host protein–carbohydrate
interactions as the basis of important infections. Adv Exp Med
Biol 491: 431–443.
Keilbaugh SA, Shin ME, Banchereau RF et al. (2005) Activation of
RegIIIb/g and interferon g expression in the intestinal tract of
SCID mice: an innate response to bacterial colonisation of the
gut. Gut 54: 623–629.
Kiers JL, Nout MJ, Rombouts FM, van Andel EE, Nabuurs MJ &
van der Meulen J (2006) Effect of processed and fermented
soyabeans on net absorption in enterotoxigenic Escherichia
coli-infected piglet small intestine. Br J Nutr 95: 1193–1198.
Kos B, Suskovic J, Vukovic S, Simpraga M, Frece J & Matosic S
(2003) Adhesion and aggregation ability of probiotic strain
Lactobacillus acidophilus M92. J Appl Microbiol 94: 981–987.
Larsen N, Nissen P & Willats WG (2007) The effect of calcium
ions on adhesion and competitive exclusion of Lactobacillus
ssp. and E. coli O138. Int J Food Microbiol 114: 113–119.
Lee YK & Puong KY (2002) Competition for adhesion between
probiotics and human gastrointestinal pathogens in the
presence of carbohydrate. Br J Nutr 88 (suppl 1), S101–S108.
Macpherson AJ & Harris NL (2004) Interactions between
commensal intestinal bacteria and the immune system. Nat
Rev Immunol 4: 478–485.
Moucadel V, Soubeyran P, Vasseur S, Dusetti NJ, Dagorn JC &
Iovanna JL (2001) Cdx1 promotes cellular growth of epithelial
intestinal cells through induction of the secretory protein PAP
I. Eur J Cell Biol 80: 156–163.
Nabuurs MJ, Hoogendoorn A, van Zijderveld FG & van der Klis
JD (1993) A long-term perfusion test to measure net
absorption in the small intestine of weaned pigs. Res Vet Sci 55:
108–114.
Neeser JR, Granato D, Rouvet M, Servin A, Teneberg S & Karlsson
KA (2000) Lactobacillus johnsonii La1 shares carbohydratebinding specificities with several enteropathogenic bacteria.
Glycobiology 10: 1193–1199.
Niewold TA, Kerstens HH, van der Meulen J, Smits MA & Hulst
MM (2005) Development of a porcine small intestinal cDNA
micro-array: characterization and functional analysis of the
FEMS Immunol Med Microbiol 54 (2008) 215–223
response to enterotoxigenic E. coli. Vet Immunol
Immunopathol 105: 317–329.
Niewold TA, Veldhuizen EJ, van der Meulen J, Haagsman HP, de
Wit AA, Smits MA, Tersteeg MH & Hulst MM (2007) The
early transcriptional response of pig small intestinal mucosa to
invasion by Salmonella enterica serovar typhimurium DT104.
Mol Immunol 44: 1316–1322.
Pretzer G, Snel J, Molenaar D, Wiersma A, Bron PA, Lambert J, de
Vos WM, van der Meer R, Smits MA & Kleerebezem M (2005)
Biodiversity-based identification and functional
characterization of the mannose-specific adhesin of
Lactobacillus plantarum. J Bacteriol 187: 6128–6136.
Reid G & Burton J (2002) Use of Lactobacillus to prevent
infection by pathogenic bacteria. Microbes Infect 4: 319–324.
Rodenburg W, Bovee-Oudenhoven IM, Kramer E, van der Meer
R & Keijer J (2007) Gene expression response of the rat small
intestine following oral Salmonella infection. Physiol Genomics
30: 123–133.
Saunders DR & Sillery J (1982) Effect of lactate and H1 on
structure and function of rat intestine. Dig Dis Sci 27: 33–41.
Sonnenburg JL, Chen CT & Gordon JI (2006) Genomic and
metabolic studies of the impact of probiotics on a model gut
symbiont and host. PLoS Biol 4: 2213–2226.
Troost F, Kleerebezem M, De Vos WM & Brummer R-JM (2006)
Identification of the acute effects of Lactobacillus plantarum
WCFS1 on transcriptional responses in human intestinal
mucosa. Gastroenterology 130: A610.
Tusher VG, Tibshirani R & Chu G (2001) Significance analysis of
microarrays applied to the ionizing radiation response. Proc
Natl Acad Sci USA 98: 5116–5121.
Unno M, Yonekura H, Nakagawara K et al. (1993) Structure,
chromosomal localization, and expression of mouse reg genes,
reg I and reg II. A novel type of reg gene, reg II, exists in the
mouse genome. J Biol Chem 268: 15974–15982.
Vasseur S, Folch-Puy E, Hlouschek V et al. (2004) p8 Improves
pancreatic response to acute pancreatitis by enhancing the
expression of the anti-inflammatory protein pancreatitisassociated protein I. J Biol Chem 279: 7199–7207.
Wold AE, Thorssén M, Hull S & Edén CS (1988) Attachment of
Escherichia coli via mannose- or Gala1 ! 4Galb-containing
receptors to human colonic epithelial cells. Infect Immun 56:
2531–2537.
Yang YH, Dudoit S, Luu P, Lin DM, Peng V, Ngai J & Speed TP
(2002) Normalization for cDNA microarray data: a robust
composite method addressing single and multiple slide
systematic variation. Nucleic Acids Res 30: e15.
2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c