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Reorganisation of the caecal extracellular matrix upon infection - relation between bacterial invasiveness and
expression of virulence genes
Angela Berndt, Jens Müller, Laura Borsi, Hartwig Kosmehl, Ulrich Methner, Alexander Berndt
To cite this version:
Angela Berndt, Jens Müller, Laura Borsi, Hartwig Kosmehl, Ulrich Methner, et al.. Reorgani- sation of the caecal extracellular matrix upon infection - relation between bacterial invasiveness and expression of virulence genes. Veterinary Microbiology, Elsevier, 2008, 133 (1-2), pp.123.
�10.1016/j.vetmic.2008.06.025�. �hal-00532449�
Accepted Manuscript
Title: Reorganisation of the caecal extracellular matrix upon Salmonella infection - relation between bacterial invasiveness and expression of virulence genes
Authors: Angela Berndt, Jens M¨uller, Laura Borsi, Hartwig Kosmehl, Ulrich Methner, Alexander Berndt
PII: S0378-1135(08)00251-4
DOI: doi:10.1016/j.vetmic.2008.06.025
Reference: VETMIC 4082
To appear in: VETMIC
Received date: 28-4-2008 Revised date: 16-6-2008 Accepted date: 26-6-2008
Please cite this article as: Berndt, A., M¨uller, J., Borsi, L., Kosmehl, H., Methner, U., Berndt, A., Reorganisation of the caecal extracellular matrix upon Salmonella infection - relation between bacterial invasiveness and expression of virulence genes, Veterinary Microbiology (2007), doi:10.1016/j.vetmic.2008.06.025
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Accepted Manuscript
Reorganisation of the caecal extracellular matrix upon Salmonella infection - relation between
1
bacterial invasiveness and expression of virulence genes
2
3
Angela Berndt a, Jens Müller b, Laura Borsi c, Hartwig Kosmehl d, Ulrich Methner b, Alexander
4
Berndte
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6 7 8 9
aInstitute of Molecular Pathogenesis, Friedrich-Loeffler-Institut, D-07743 Jena, Germany
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b Institute of Bacterial Infections and Zoonoses, Friedrich-Loeffler-Institut, D-07743 Jena, Germany
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cIstituto Nazionale per la Ricerca sul Cancro, 16132 Genoa, Italy
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dInstitute of Pathology, HELIOS-Klinikum, D-99012 Erfurt, Germany
13
eInstitute of Pathology, Friedrich Schiller University, D-07740 Jena, Germany
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15 16 17 18 19 20 21 22 23
Correspondence to:
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Dr. Angela Berndt, Friedrich-Loeffler-Institut, Institute of Molecular Pathogenesis, Naumburger
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Strasse 96a, D-07743 Jena, Germany, Phone: ++49 3641 804 410,
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Fax: ++49 3641 804 228, e-mail: angela.berndt@fli.bund.de
27
Manuscript
Accepted Manuscript
Abstract
1
Interactions of Salmonella (S.) outer membrane structures with extracellular matrix (ECM) of host
2
tissues seem to be crucial for bacterial adhesion and invasion. To evaluate the relationship between the
3
ECM and bacterial invasiveness, the reorganisation of fibronectin, tenascin-C and laminin after
4
Salmonella exposurein vivo, the Salmonellaadhesiveness to ECM proteinsin vitroand the virulence
5
gene expression upon co-cultivation of salmonellae and ECM proteins were elucidated for two
6
Salmonella strains with different capabilities to enter the intestinal mucosa. Immunohistochemistry
7
and confocal microscopy showed that the infection of day-old chicks using either the highly invasive
8
S. Enteritidis (SE) or the nearly non-invasive S. Infantis (SINF) strain was associated with an invasion-
9
dependent reorganisation of fibronectin and tenascin-C in the caecal wall. Compared to SINF,
10
clustered formations of SE were localised within and attached to the fibronectin and tenascin-C
11
scaffold in the lamina propria indicating a relevance of ECM for bacterial dissemination in lower
12
regions of the mucosa. In adhesion assays, SE was, indeed, significantly more adhesive to the matrix
13
proteins than SINF. The attachment was accompanied by an increased fliC mRNA expression in SE
14
demonstrated by microarray analysis as well as quantitative real-time RT-PCR. The data suggest a
15
relationship between the capability of Salmonella serovars to interact with matrix proteins and to
16
disseminate in gut mucosa perhaps in consequence of a matrix-mediated upregulation of the
17
Salmonella motility gene fliC.
18 19 20 21 22
Key words: Salmonella, chicks, extracellular matrix, virulence gene
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Accepted Manuscript
Introduction
1
Human gastroenteritis caused bySalmonella(S.)enterica ssp. enterica infection represents one of the
2
most important food-born zoonosis worldwide. The main sources for Salmonella infections in humans
3
are eggs, egg products as well as poultry meat products. In spite of comprehensive preventions and
4
control measures against Salmonella in poultry industries, human food poisoning by Salmonella
5
enterica spp. is continuing to be a major public health problem. Therefore, basic research on
6
immunological mechanisms responsible for Salmonella adhesion, invasion, colonisation and
7
persistence in birds is of great interest especially to develop more efficient vaccines in future. Upon
8
Salmonella infection of birds, a range of immune mechanisms are initiated to eradicate Salmonella
9
organisms entering the gut mucosa (Van Immerseel et al., 2005). Beside a found protective relevance
10
of some special immune cell subsets (Berndt et al., 2006), there is increasing evidence that also
11
acellular components, such as proteins of the extracellular matrix (ECM), are involved in immunity as
12
well as inflammation (Fiocchi, 1997). Thus, the immune answer seems to be associated with
13
sophisticated immune-non-immune cell interactions, which occur in the midst of a complex mixture of
14
matrix proteins including fibronectin, collagen, laminin or tenascin (Raghow, 1994). These acellular
15
components of the extracellular matrix can play an active role in immune regulation under both
16
normal and inflammatory conditions (Raghow, 1994; Fiocchi, 1997). This includes not only
17
degradation of pre-existing matrix protein structures but also the de novo synthesis of adhesion protein
18
variants, especially of laminin, fibronectin and tenascin-C, normally occurring during embryogenesis.
19
Finally, a so-called provisional ECM will be formed (Kosmehl et al., 1996). The structural
20
modification of the extracellular matrix is highly regulated and mediated by inflammatory cells,
21
endothelial cells, fibroblasts as well as epithelial cells. The newly formed ECM provides spatially
22
defined differentiation, proliferation and migration signals, which promote immune reactions, but may
23
be crucial also for bacterial dissemination in epithelial surfaces as the mucosal wall.
24
Extremely limited information exists on the amount, organisation and function of ECM in intestinal
25
inflammation, particularly with regard to avian salmonellosis. However, the interaction of Salmonella
26
outer membrane structures with components of the host extracellular matrix may be a crucial
27
prerequisite for mucosal attachment, colonisation and invasion. For S. Enteritidis, the capability to
28
adhere to extracellular matrix proteins such as fibronectin, laminin or collagen (Kukkonen et al., 1993)
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Accepted Manuscript
has been described. The functional importance of this interaction has not been completely understood
1
yet, but seems to be a prerequisite for intestinal colonisation and persistence at areas of epithelial
2
erosion or at the luminal surface of intestinal epithelial cells.
3
In the presented study, we compared two Salmonella serovars (S. Enteritidis as a highly invasive and
4
S. Invantis as a hardly invasive strain; for details of the strains see Berndt et al., 2007) concerning their
5
impact on the reorganisation of the ECM in caecum as well as their adhesion potential to different
6
extracellular matrix proteins in connection with the resultant virulence gene expression. At first,
7
structural changes of fibronectin, laminin, and tenascin-C matrix were examined in caecum of non-
8
treated compared to S. Enteritidis (SE) and S. Infantis (SINF) infected day-old chicks using
9
immunohistochemistry. Secondly, the spatial distribution of the Salmonella organisms in relation to
10
the matrix protein scaffold was determined by means of confocal laser-scanning microscopy. Thirdly,
11
the two differently invasive Salmonella strains were evaluated concerning their ability to bind ECM
12
components, such as fibronectin, laminin and basement membrane (BM) matrix and to express various
13
virulence genes by an in vitroadhesion assay and microarray analysis, respectively.
14 15
Materials and Methods
16
Animals
17
Specific pathogen-free (SPF) White Leghorn chickens were hatched at the facilities of the Friedrich
18
Loeffler Institute (Jena, Germany) from eggs obtained from Charles River Deutschland GmbH
19
(Extertal, Germany). Experimental and control (non-treated) groups were kept in separate rooms;
20
commercial feed (in powder form without antibiotics or other additives) and drinking water were both
21
available ad libitum. Cleaning and feeding regimens were organised, which effectively prevented
22
cross-contamination throughout the experiment. The animal experiment was performed in accordance
23
with the German Animal Protection Act (registration number: 04-01/04)
24
25
Bacterial strains and experimental design
26
Newly hatched day-old chicks were infected orally using the Salmonella serovars Enteritidis 147 (SE)
27
and Infantis 1326 (SINF). The bacterial strains were selected from a pool of Salmonella-strains as
28
typical representatives of the respective serovar (for detailed information on the strains see Methner et
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Accepted Manuscript
al., 2006; Berndt et al., 2007
)
. Former studies in our lab proved that SE is highly invasive and a strong1
immune stimulator, whereas SINF displays hardy any invasiveness and is only a slight elicitor of any
2
immune reaction in gut. Nevertheless, both serovars are able to colonise the gut lumen by similar high
3
bacterial cell counts (Berndt et al., 2007). These two Salmonella strains were now used for the
4
presented study. Storage, cultivation and application of the strains were performed as described
5
(Berndt et al., 2007). After cultivation of the bacteria suspensions, doses were adjusted (1-2 x 107 cfu
6
per bird in 0.1 ml) and one-day old chicks infected orally (0.1 ml/bird) as described (Berndt and
7
Methner, 2001). Three animals per investigation day and group (SE-infected, SINF-infected and non-
8
treated control group) were examined 2, 4 and 9 days after infection (days 3, 5 and 10 of life,
9
respectively). For this purpose, animals were sacrificed and caecum samples taken as described
10
(Berndt et al., 2007). Caecum samples were shock frozen and stored in liquid nitrogen.
11 12
Immunohistochemistry
13
Changes in total fibronectin (tFn), extradomain A containing cellular fibronectin (EDA+Fn), total
14
laminin (Ln), laminin-332 (Ln-332; formerly laminin-5; a component of the hemidesmosomes of the
15
epithelial BM), and total tenascin-C (Tn-C) expression pattern were analysed 2, 4 and 9 days SE and
16
SINF infection in caecum by immunohistochemistry (3 animals per group and day). The primary
17
monoclonal and polyclonal antibodies, reactive against chicken fibronectin variants (tFn; EDA+Fn [a
18
kindly gift from Prof. Zardi and Dr. Borsi, Genova, Italy]), laminin variants (Ln; Ln-332 [clone R14, a
19
kindly gift from Prof. Aumailly, Cologne, Germany]), tenascin-C (Tn-C;) and Salmonella common
20
antigen (LPS) were used. For immunohistochemistry, cryostat sections (7 µm in thickness) of the
21
respective frozen caecum samples were fixed in ice cold acetone for 15 minutes. Non-specific staining
22
due to endogenous biotin was inhibited applying the DAKO Biotin Blocking System according to the
23
manufacturer’s instructions (DakoCytomation, Hamburg, Germany). After that, the primary antibody
24
was incubated at 4 °C over night. Immunohistochemical staining was performed using the DAKO
25
REALTM Detection System (Alkaline Phosphatase/Red; Rabbit/Mouse; DakoCytomation, Hamburg,
26
Germany) according to the manufacturer’s protocol. Sections were counterstained with haematoxylin.
27
As negative control, the antibodies were replaced by non-immune serum.
28
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Confocal laser scanning microscopy (CLSM)
1
For co-localisation analysis of extracellular matrix proteins and Salmonella organisms by CLSM,
2
acetone-fixed cryostat sections (10 µm thick) of caecum samples of all animals (3 per group and day)
3
were first incubated with the primary antibody against Salmonella (Tab. 1). This antibody was
4
detected using an Alexa-488-conjugated goat-anti-mouse-IgG2a immunoglobulin (Invitrogen,
5
Karlsruhe, Germany). Subsequently, sections were incubated with the polyclonal rabbit antibodies
6
against fibronectin, laminin or tenascin-C and detected using a Cy3-conjugated goat-anti-rabbit
7
immunoglobulin (DAKO). In the case of combination of the monoclonal Salmonella antibody with the
8
monoclonal EDA+Fn antibody IST-9, the detection was realised using the isotype-specific anti-mouse-
9
IgG2a antibody conjugated with Alexa-488 (Invitrogen) and the isotype-specific anti-mouse-IgG1
10
antibody conjugated with Alexa-555 (Invitrogen), respectively. Finally, the sections were washed and
11
mounted with glycerine gelatine. Fluorescence labelling was analysed by confocal laser-scanning
12
microscopy (LSM 510, Zeiss, Germany). Simultaneous detection of fluorescence emission using an
13
argon (488 nm) and HeNe laser (543 nm) in combination with the light filters BP 505-530 and LP 560
14
(Zeiss, Germany) resulted in a two channel image: Channel 1 represented Cy3 or Alexa-555
15
fluorescence (displayed as red) and Channel 2 represented Alexa-488 (displayed as green).
16 17
Adhesion assay
18
To analyse the capability of the Salmonella serovars Enteritidis (SE) and Infantis (SINF) to attach
19
proteins of the extracellular matrix 12 well TC cell culture plates (Greiner Bio-One GmbH,
20
Frickenhausen, Germany) were coated with 10 µg/cm2 of recombinant human cellular fibronectin
21
containing the EDA domain (EMP Genetech, Ingolstadt, Germany), laminin from Engelbreth-Holm-
22
Swarm (EHS) mouse tumour and MatrigelTMBasement Membrane Matrix from EHS mouse tumour
23
(both from BD Biosciences, Heidelberg, Germany). For this, 400 µl D-PBS without calcium or
24
magnesium (Invitrogen GmbH, Karlsruhe, Germany) but containing 40 µg of the respective matrix
25
protein was added to each well and incubated for 90 min at room temperature. After washing the plate
26
twice with D-PBS, 4.5 x 106 bacteria (SE or SINF) in 3 ml PBS (without calcium or magnesium) were
27
applied to each well and cultivated for 2 h at 41 °C. After careful rinsing twice with PBS (41 °C), 1 ml
28
ice-cold PBS was added to each well and adherent bacteria were detached using a disposable cell
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Accepted Manuscript
scraper (Greiner Bio-One GmbH). The number of attached bacteria was determined by dilution of the
1
bacterial suspension between 1:100 and 1:1,000,000 and spreading on xylose lysine desoxycholate
2
(XLD) agar plates (Merck, Hamburg, Germany). The agar plates were incubated for 18 2 h at 41 °C.
3
After incubation, the Salmonella colonies were counted and the total number of adherent bacteria per
4
well was calculated. Two experiments were performed in triplicate.
5 6
Salmonella-matrix co-cultivation and microarray-based virulence gene expression analysis
7
To analyse virulence gene expression by the Salmonella serovars in consequence of matrix protein
8
contact 1-2 x 107 bacteria per ml of SE and SINF were cultivated in M9 minimal medium with 0.5 %
9
glucose. After 4 h, recombinant human cellular fibronectin (final concentration 30 µg/ml; EMP
10
Genetech, Ingolstadt, Germany), laminin from EHS mouse tumour (final concentration 30 µg/ml; BD
11
Biosciences, Heidelberg, Germany), and MatrigelTM Basement Membrane Matrix from EHS mouse
12
tumour (final concentration 45 µg/ml; BD Biosciences) were added to the separate bacterial cultures.
13
After further 4 h of cultivation, the bacteria were collected by centrifugation (5 min, 5000 rpm) and
14
total RNA was extracted for microarray experiments as described below. Bacterial cultures without
15
matrix proteins or supplemented only with the dilution buffer were used as controls. The test was
16
performed in triplicate.
17
After co-cultivation, the transcriptional activity of 46 virulence-associated genes of SE and SINF
18
(Tab. 2) was analysed separately for each test by means of the ArrayTubeTM microarray system
19
(Clondiag Chip Technologies, Jena, Germany). Two hybridisation probes for each gene located near
20
3'- and 5'-ends of the open reading frame were designed using Vector NTI Software (Invitrogen,
21
Karlsruhe, Germany) on the basis of a genome sequence of S. Enteritidis available from the Wellcome
22
Trust Sanger Institute (http://www.sanger.ac.uk/Projects/Salmonella). Probe sequences are available
23
upon request. Oligonucleotides were purchased as 3'-amino-modified oligos from Metabion
24
(Martinsried, Germany). The probes were spotted on the ArrayTubeTM in double replication. The
25
spotting procedure was recently described (Sachse et al., 2005).
26
For hybridisation, total bacterial RNA of each test was extracted using the RNeasy Bacteria Protect
27
Mini Kit (Qiagen, Hilden, Germany) with additional DNAse treatment according to the instructions of
28
the manufacturer. Quality and quantity of RNA was determined by UV spectrophotometry. In each
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Accepted Manuscript
case, 10 µg of total RNA was reverse transcribed and labelled with biotin using the LabelStar Array
1
Kit (Qiagen) according to the manufacturer's instructions. Evaluation of quality of the array probes
2
was done by hybridisation with labelled Salmonella DNA. For that, DNA of SE and SINF was
3
extracted using a DNeasy – Kit (Qiagen) and labelled using the Biotin DecaLabel DNA Labeling Kit
4
(Fermentas GmbH, St. Leon-Rot, Germany) according to the instructions of the manufacturer.
5
The hybridisation procedure and analysis of the results were performed as described (Sachse et al.,
6
2005). After a conditioning of the ArrayTubeTM, the hybridisation was carried out at 50 °C for 60 min.
7
The hybrids were stained using Poly-HRP-Streptavidin (Perbio Science, bonn, Germany), peroxidase
8
and o-Dianisidine substrate solution SeramunGrün (Seramun Diagnostica GmbH, Heidesee,
9
Germany). The results of hybridisation were monitored by the ATR-01 array tube reader (Clondiag
10
Chip Technologies).
11 12
Quantitative real-time RT-PCR
13
To analyse and confirm the influence of the matrix proteins on fliC (the only one significantly
14
increased in microarray analysis after co-cultivation with two matrix proteins; see results) mRNA
15
expression of SE and SINF a quantitative real-time RT-PCR was performed in duplicate using the
16
total bacterial RNA extracted for the three array experiments. Amplification and detection of specific
17
products were performed using the Mx3000PTM real-time PCR equipment (Stratagene, La Jolla, CA)
18
using the following temperature-time profile: one cycle of 50 °C for 30 min and 95 °C for 15 min, 45
19
cycles of 94 °C for 30 s, respective annealing temperature for 30 s (16S: 57 °C; SE fliC: 56 °C; SINF
20
fliC: 57 °C), followed by 72 °C for 30 s. Primer sequences were 16S-F: 5’-
21
ACTTGGAGGTTGTGCCCTTGAG-3’, 16S-R: 5’-GCCCCCGTCAATTCATTTGA-3’ (accession
22
number: U90318); SE fliC-F: 5’-AATCAATGAAGACGCTGCCG-3’, SE fliC-R: 5’-
23
TGAATTGCCCCCAGAGAAGA-3’ (accession number: M84980); SINF fliC-F: 5’-
24
ACGCTGCAAGTAAAGCCGAAG-3’, SINF fliC-R: 5’-GTGTCAACCTGTGCCAAAGCA-3’
25
(accession number: DQ095521). To check the specificity of the amplification products, the
26
dissociation-curve method was used (one cycle at 95 °C for 1 min, 55 °C for 30 s, and 95 °C for 30 s)
27
subsequent to amplification. The threshold method was used for quantification of the mRNA level.
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ΔCT (cycle threshold change) values were calculated on the basis of the internal standard 16S. The
1
results were expressed as 2(-ΔΔCT) (n-fold change).
2 3
Statistical analysis
4
Viable counts of bacteria were shown as percentages of attached bacteria and differences between the
5
groups were statistically analysed by the Mann-Whitney-Wilcoxon-test. The data of the microarray
6
and quantitative real-time RT-PCR were analysed by means of the Student’s-t -test. In the case that the
7
given P-values were equal or less than 0.05, there was a statistically significant difference at the
8
95.0 % confidence level in both tests. All confidence levels were given with the data (*: P 0.05; **:
9
P 0.01).
10 11
Results
12
Fibronectin expression after Salmonella infection
13
To investigate the occurrence and distribution of the matrix protein fibronectin in caecum of chicks
14
either infected with the highly invasive Salmonella serovar Enteritidis (SE) or the nearly non-invasive
15
serovar Infantis (SINF) total fibronectin (tFn) was immunohistochemically detected using a polyclonal
16
rabbit antibody and the EDA+ splicing variant of cellular fibronectin (EDA+Fn) by means of the
17
monoclonal antibody IST-9 directed against the extra domain A (Table 1).
18
tFn was found widely distributed over the lamina propria, submucosa and tunica muscularis (Figures
19
1A-C) in caecum of all animals, independently of age or treatment. The epithelial lining was regularly
20
negative, with a few positive cells in SE infected animals at times. Interestingly, the staining pattern of
21
EDA+Fn changed in the course of normal development of non-treated animals. In newly hatched and
22
non-treated chicks, EDA+Fn was detected in the tunica muscularis and lamina propria of caecum and
23
focused on the stromal tips of the villi. In the mucosa and submucosa of 10 day-old chicks, the amount
24
of EDA+Fn was noticeably reduced (Figures 1D and 2A-C).
25
Compared to non-treated animals, a strong increase of EDA+Fn was seen after infection with the
26
highly invasive SE strain in stroma of the villi and the submucosa. Within the lamina propria, EDA+Fn
27
deposition was characterised by a stromal network-like pattern (Figure 1E). Additionally, numerous
28
EDA+Fn positive but tenascin-C and laminin negative cells were localised in caecal epithelium
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(Figures 2D and E). Luminal depositions of tFn or EDA+Fn by epithelial cells have never been
1
observed during the course of the SE infection.
2
The infection with the low invasive SINF led to an increased amount of EDA+Fn in caecal mucosa,
3
but on a lower level, which was probably caused by the lesser extended swelling of the caecal villi in
4
SINF- compared to SE-treated birds. (Figure 1F).
5
Similar to non-treated animals, a reduction of EDA+FN positively stained mucosal areas was seen at
6
day 9 after infection in both SE and SINF infected birds.
7 8
Tanascin-C expression in caecum afterSalmonella infection
9
To investigate the occurrence and distribution of all variants of tenascin-C (Tn-C) in caecum of non-
10
treated and Salmonella (SE and SINF) infected animals a polyclonal rabbit antibody was used (see
11
Table 1).
12
During the early caecal development of newly hatched chickens, Tn-C was localised in the tunica
13
muscularis as well as the lamina propria and focused on the stromal tips of the villi. Additionally,
14
there was a strong positive staining of the submucosa (Figure 1G). In contrast to EDA+Fn, changes of
15
the Tn-C expression were never seen during the first 10 days of life of non-treated birds.
16
After infection with the highly invasive Salmonella strain SE, an apparent increase of Tn-C deposition
17
was found in the lamina propria as well as the submucosa and characterised by a typical stromal
18
network-like pattern. The infection with the lower invasive Salmonella strain SINF led to an increased
19
quantity of Tn-C similar to that seen upon SE infection in caecal mucosa and submucosa, but to a
20
lower degree which was probably caused by the lesser extended swelling of the caecal villi in SINF-
21
compared to SE-treated birds. Luminal depositions of Tn-C by epithelial cells have never been seen,
22
neither after SE nor SINF infection. (Figures 1G-I).
23 24
Laminin deposition and immunohistochemical basement membrane integrity upon Salmonella
25
infection
26
Laminins are a family of heterotrimeric proteins consisting of differently large () and small (and )
27
chains. Laminin was detected immunohistochemically using a polyclonal rabbit antibody detecting all
28
Accepted Manuscript
variants (Ln). Additionally, we used a polyclonal antiserum against laminin-332, a laminin variant
1
localized in the epithelial basement membrane as a component of the hemidesmosomes (Table 1).
2
The antibody against all laminin variants regularly stained the epithelial basement membrane, blood
3
vessel structures in the lamina propria and submucosa as well as the lamina muscularis mucosae and
4
the tunica muscularis of non-treated chicks. After infection with SE or SINF, the deposition pattern
5
was not changed and Ln re-deposition in inflammatory areas, as seen for EDA+Fn or Tn-C, was not
6
evident (Figures 1K-M).
7
The laminin variant Ln-332 was detected in the epithelial basement membrane (BM) of the villi but
8
not the crypts of non-treated animals. The staining intensity decreased continuously from the tip to the
9
base of the villi. Additionally, there was a slight positive Ln-332 staining in the lamina muscularis
10
mucosae. After infection with SE or SINF, Ln-332-associated BM alterations were not seen (Figure
11
12
2F).13
Co-localisation of Salmonella and matrix proteins
14
To show a possible attachment of SE and SINF organisms to extracellular matrix structures in caecum
15
double-immunofluorescence technique and confocal laser-scanning microscopy were used.
16
In general, SE was highly invasive (Figures 2G-I), while SINF was nearly non-invasive and hardly
17
able to enter lower regions of the caecal mucosa (Figures 2K-M). Four days after infection, larger
18
clusters of SE were found in the stromal compartment of the villi. These SE clusters were situated
19
within the meshes of both the EDA+Fn and tenascin-C matrix network, sometimes overlapping the
20
scaffold structures. (Figure 2G and I). Occasionally, low numbers of SINF were detected within the
21
meshes of the Tenascin-C or EDA+Fn matrix (Figure 2K and M). For the laminin structures in the
22
lamina propria and epithelial basement membrane, such an association has never been observed
23
(Figure 2H and L). Furthermore, differences in fluorescence intensities between the two infected
24
animal groups indicated an enhanced EDA+Fn deposition in SE compared to SINF treated chicks
25
(Figures 2G and K).
26
27
28
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Adhesiveness of the Salmonella serovars to cellular fibronectin, laminin and MatrigelTM
1
To examine the capacity of SE and SINF to attach to recombinant cellular Fn containing the EDA
2
domain (EDA+Fn) as well as to laminin and MatrigelTM (basement membrane matrix from EHS mouse
3
tumour) an in vitroadhesion assay was used. As shown in figure 3A, SE exhibited generally a higher
4
adhesiveness to all matrix proteins than SINF (P 0.05). Among the proteins used for surface coating,
5
cellular Fn was the most potent matrix component for SE immobilisation (comparison Fn to
6
MatrigelTM: P 0.05). In contrast, SINF showed lowest adhesiveness to Fn.
7 8
Influence of cellular fibronectin, laminin and MatrigelTM on virulence gene transcription of the
9
Salmonella serovars
10
To analyse the influence of the matrix proteins on the transcription of different Salmonella virulence
11
genes the two Salmonella strains SE and SINF were co-cultivated with the ECM proteins (cellular
12
fibronectin, laminin, MatrigelTM) for 4 h. Subsequently, a Salmonella microarray was performed. The
13
test was done in triplicate.
14
Prior the study, the quality of the array probes was evaluated by hybridisation of the microarray with
15
Salmonella(SE, SINF) DNA samples (data not shown). After hybridisation of DNA from SE, all spots
16
showed strong signals indicating sequence complementarities between probes and sample DNA. The
17
hybridisation of DNA from SINF resulted in a low number of spots with weak or no signals signifying
18
single to multiple mismatches between the immobilized probes and the sample DNA. The SINF genes
19
fliC,sodC1 and spvB were not detectable in SINF.
20
After cultivation of SE and SINF in presence of cellular fibronectin, laminin or MatrigelTM, only a few
21
genes showed changes in their transcriptional levels. Notably, the fliC expression rate of SE was
22
clearly upregulated after co-cultivation with laminin, MatrigelTM(P 0.01) as well as to a lower
23
degree with fibronectin (P 0.05). Additionally, some differences were seen between the two
24
Salmonella serovars.
25
In case of fibronectin, 14 of 46 analysed SE virulence genes exhibited changes in mRNA expression
26
(Figure 4A). There was a definite increase in transcriptional activity of the genesfliC, phoP, and pspA.
27
The SE genes avrA, relA, lon, sodC1, and clpPexhibited only a moderately increased transcriptional
28
Accepted Manuscript
activity in presence of fibronectin. The mRNA expression rate of the genes ssaN, sseA, rpoS, spoT,
1
and osmCwas downregulated.
2
In the case of SINF cultivated in the presence of fibronectin, eight genes (avrA, phoP, rpoS, relA, lon,
3
clpP, osmC, and pspA) revealed similar transcriptional levels as found in SE. While the SPI-2 genes
4
ssaN, sseA, and ssaBas well as the regulatory gene spoT were transcriptionally upregulated in SINF,
5
these genes were inhibited or remained unchanged (ssaB) in SE. The mRNA expression activity of
6
sodC1has never been found in SINF.
7
After Salmonella co-cultivation with 5 % (w/v) MatrigelTMbasement membrane matrix, 16 genes of
8
both SE and SINF changed their transcriptional activity (Figure 4B). The mRNA expression levels of
9
the genes avrA, ssaN, sseA, ssaB, phoP, relA, spoT, clpP, and pspA did not show significant
10
differences between the two serovars. The copy numbers of ssaV, ssrA, sodC1, rpoS, and lon were
11
increased in SE and down-regulated (rpoS, lon) or stable (ssaV, ssrA, sodC1) in SINF. Furthermore,
12
the transcriptional activity of osmC was not detectable in SE, but decreased in SINF. The strongest
13
Matrigel-related upregulation was detected in SE for the gene fliC (up to 500 %).
14
The impact of laminin on SE and SINF resembled that of MatrigelTM basement membrane matrix
15
(Figure 4C). In contrast to the MatrigelTM effect, the transcriptional levels of the genes rpoS and osmC
16
did not change in the presence of laminin.lonactivity has never been detected in SINF. Transcription
17
of fliC in SE was up-regulated by more than 500 %.
18 19
fliCmRNA expression of S. Enteritidis and S. Infantis upon matrix protein co-cultivation
20
To substantiate the increased fliC mRNA expression found by microarray analysis and to show ECM-
21
dependent regulation of this gene in SINF quantitative real-time RT-PCR was performed. The RT-
22
PCR analysis confirmed the array expression data of the up-regulation of fliC mRNA in SE after
23
interaction with the ECM components. The most prominent changes were seen after interaction with
24
laminin (P 0.05) followed by cellular fibronectin (P 0.05) and MatrigelTM (P 0.05). In contrast to
25
that, SINF showed neither a significantly increased nor decreasedfliCexpression (Figure 3B).
26
27
28
29
Accepted Manuscript
Discussion
1
The presented study is a detailed report on fibronectin (Fn), tenascin-C (Tn-C) and laminin (Ln)
2
reorganisation upon Salmonella infection (SE, SINF) of newly hatched chicks with respect to the
3
spatial distribution of bacteria in gut mucosa. The matrix proteins examined exist in different isoforms
4
generated by alternative splicing (Fn, Tn-C) or alternative chain assembly (Ln). The isoforms reveal
5
various biological properties and are differently expressed during embryogenesis and physiological or
6
pathological tissue remodelling (Kosmehl et al., 1996).
7
During the regular gut development of non-treated newly hatched chicks of our study, tFn, EDA+Fn
8
and Tn-C were abundantly expressed in the lamina propria. This result is in line with other reports on
9
the high and characteristic expression of Fn and Tn-C splice variants during the embryonic or juvenile
10
gut morphogenesis of birds and humans (Beaulieu et al., 1991; Tucker et al., 1994).
11
After Salmonella infection of the day-old chicks, a strong increase of EDA+Fn and Tn-C depositions
12
was observed especially pronounced in the case of the highly invasive Salmonella strain SE. Similar,
13
an upregulation of these both proteins has been described for several other inflammatory conditions,
14
such as septic responses, bacterial infections, glomerulopathies and fibroses (Assad et al., 1993;
15
Päällysaho et al., 1993; Satoi et al., 2000). The significance of this phenomenon has not been
16
completely understood yet. On the one hand, infection-associated ECM reorganisation may be a
17
defence strategy of the host and facilitate the immune response against salmonellae. Indeed, the EDA
18
domain of fibronectin is able to activate the toll-like receptor 4 (TLR4) that constitute a signalling
19
receptor of the innate immunity known to be triggered by LPS (Okamura et al., 2001). Furthermore,
20
EDA+Fn can stimulate dendritic as well as mast cells and induce cytotoxic T cell responses
21
(Gondokaryono et al., 2007; Lasarte et al., 2007) with the latter being of central importance for the
22
defence against Salmonella infections in chickens (Berndt and Methner, 2001).
23
On the other hand, the inflammatory ECM reorganisation might be of pathophysiological importance
24
in the course of Salmonella infections and assist the bacteria by enhancing their adhesion to and
25
penetration of cellular structures or by modulation of virulence gene expression. Indeed, the adhesion
26
of Salmonella and several other bacteria to different extracellular matrix proteins has already been
27
reported and postulated as a prerequisite for colonisation and invasion of pathogens (Secott et al.,
28
2002; Medina, 2004). However, a deposition of matrix proteins at the luminal side of the epithelial
29
Accepted Manuscript
lining, which naturally constitute the first place of Salmonella attachment in gut, has never been found
1
in infected animals of our study. Instead, we observed a spatial association of SE with the scaffold of
2
EDA+Fn and Tn-C in the lamina propria suggesting a role of these matrix proteins for bacterial
3
dissemination at least within lower regions of the intestinal mucosa. This hypothesis is supported by
4
our result of a generally enhanced adhesion capability of SE to ECM coated surfaces compared to
5
SINF. Thus, SE might have used its matrix binding potential in order to disseminate in the lamina
6
propria.
7
Although the adherence of Salmonella and other bacteria to laminin and fibronectin seems to be
8
associated with their colonisation and invasion (Dorsey et al., 2005), a possible effect of this
9
interaction on bacterial gene transcription activity has not been described yet. We performed for the
10
first time a microarray-based virulence gene expression analysis after contact of the highly invasive
11
SE or the nearly non-invasive SINF with cellular fibronectin in comparison to laminin and complex
12
basement membrane derived matrix MatrigelTM. In general, the presence of extracellular matrix
13
components in the Salmonella culture medium modified the transcription of several virulence genes in
14
both serovars indicating an outside-in signalling during bacteria-ECM co-cultivation. Nevertheless, the
15
changes of gene expression levels were merely moderate and the biological significance of most of
16
them has to be the subject of further studies. Notably, fliC mRNA expression exhibited regularly a
17
considerable upregulation in consequence of the interaction of SE and the matrix proteins. By
18
quantitative real-time RT-PCR, the array data of SE were confirmed and the unchanged fliC mRNA
19
expression answer of SINF evidenced. The flagella subunit protein FliC, which is found on the
20
Salmonella surface in larger quantities, serves as ligand of the TLR5 and represents a major pro-
21
inflammatory agent in vivo (Hayashi et al., 2001; Means et al., 2003). Previous studies have shown
22
that bacterial flagellin is a potent stimulator of avian heterophils (Kogut et al., 2006), which are proved
23
to be crucial in the early stages of the Salmonella infection in birds (Van Immerseel et al., 2005).
24
Others reported that flagellin is able to induce dendric cell maturation and promote the development of
25
an adaptive immune response (McSorley et al., 2002; Means et al., 2003). Indeed, a previous study of
26
our laboratory demonstrated SE as a powerful immune stimulator in vivo that triggered higher
27
cytokine gene expression rates and a more pronounced immune cell influx compared to SINF (Berndt
28
Accepted Manuscript
et al., 2007). Whether our finding of anin vitro upregulation of the fliC gene transcript upon matrix
1
protein co-cultivation might be the same in vivo must remain open.
2
That FliC plays also a crucial role for bacterial invasion into epithelial cells as well as for Salmonella
3
adhesion and colonisation in avian gut has been shown in former studies (Allen-Vercoe and
4
Woodward, 1999; Parker and Guard-Petter, 2001; La Ragione et al., 2003). Thus, Igimi et al. (2006)
5
reported a FliC-detecting antibody that prevents SE from adhering to and invading the human
6
intestinal epithelial cell line, Caco-2, and suggested that flagellin may potentially be useful as an
7
element of a Salmonella vaccine.
8
In the presented study, components of the extracellular matrix have been analysed to provide a deeper
9
insight into the role of these important structural proteins during pathogenetic processes. We were able
10
to show that Salmonella infection and invasion is associated with a reorganisation of fibronectin and
11
tenascin-C matrix in the caecal wall. The higher capacity of SE to bind proteins of the ECM as well as
12
the subsequent increased fliC mRNA expression might have been the cause for its more efficient entry
13
and dissemination in the host tissue compared to SINF. Our work can expand the knowledge about
14
both the acellular defence activities of chicks against Salmonella infection and the virulence
15
mechanisms of bacteria fine regulated after encounter with special host structures. Further studies will
16
be valuable in better understanding the interrelations and interdependencies of acellular host responses
17
and pathogenic mechanisms of bacteria. The investigation of tissue reactions and bacterial actions
18
after pathogen-host encounter may help to develop more effective Salmonella vaccines for poultry
19
industries and eventually better protect humans against Salmonella-caused food poisoning in future.
20 21
Acknowledgement
22 23
The authors would like to thank Prof. Monique Aumailley for the generous gift of the anti laminin-332
24
antibodies. The authors are grateful to Katrin Schlehahn, Susanne Bergmann and Christiane Geier for
25
excellent technical assistance. The study was partially supported by the European Community
26
(FOOD-CT-2003-505523 “SUPASALVAC”; this publication reflects only the authors view. The
27
European Commission is not liable for any use that may be made of the information contained). The
28
microarray analysis was supported by a grant of the “Akademie für Tiergesundheit e.V.” (Bonn-Bad-
29
Godesberg).
30
Accepted Manuscript
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Accepted Manuscript
Figure captions
1
Figure 1
2
Immunohistochemical detection of total fibronectin (tFn; A-C), EDA domain containing cellular
3
fibronectin (EDA+Fn; D-F), total tenascin-C (Tn; G-I) and total laminin (Ln; K-M) in caecum of
4
normal (A, D, G, K), Salmonella Enteritidis 147 (SE; B, E, H, L) and Salmonella Infantis 1326
5
(SINF; C, F, I, M) infected animals at day 5 of life or at day 4 after infection.
6
blue coloured: cell nuclei; red coloured: ECM proteins; arrow: stoma tip of the villi; arrowhead:
7
lamina muscularis mucosae; double arrow: blood vessel; short thick arrow: epithelial basement
8
membrane, L: lumen; E: epithelium; LP: lamina propria; SM: submucosa; M: tunica muscularis;
9 10
Figure 2
11
EDA domain containing cellular fibronectin (EDA+Fn) expression in the caecal wall of newly hatched
12
chickens (A), in the developing chicken gut at day 5 of life (B; arrow: stoma tip of the villi) and after
13
10 days of life (C). (D) EDA+Fn positive cells in the epithelium of S. Enteritidis infected animals are
14
indicated by the arrow. (E) The EDA+Fn positive cells are not stained for tenascin-C. (F) Ln-332
15
variant stained in a non-infected animal at day 5 of life. Only the basement membrane (arrow) and
16
lamina muscularis (LM) are positively stained (arrow: epithelial basement membrane; arrowhead:
17
crypts).
18
Double immunofluorescence immunostaining of the LPS of S. Enteritidis (G-I; green) or S. Infantis
19
(K-M; green) and EDA+Fn (G and K; red), laminin (H and L; red) and tenascin-C (I and M, red) in
20
caecum, 4 days after infection. Only S. Enteritidis showed larger clusters within the EDA+Fn and
21
tenascin-C network of the stromal compartment of the villi, which frequently overlapped with the
22
matrix scaffold (G and I). For both the laminin-positive structures and the epithelial basement
23
membrane, an association between bacteria and matrix have never been seen. (H).
24
L: lumen; E: epithelium; LP: lamina propria; LM: lamina muscularis; SM: submucosa; M: tunica
25
muscularis
26
27
Figure 3
28
A:Graphical representation of the adhesiveness of S. Enteritidis (SE) and S. Infantis (SINF) to cellular
29
fibronectin (FN) laminin (LN) and MatrigelTM given as the mean values ( standard deviation) of the
30
percentage of attached bacteria. Asterisks indicate a significant difference between the two treated (SE
31
and SINF) groups (P 0.05). The letter “a” indicates a significant difference (P 0.05) between
32
fibronectin and laminin, the letter “b” between fibronectin and Matrigel and “c” between laminin and
33
Matrigel.
34
B: Graphical representation of changes in fliC mRNA expression after interaction of S. Enteritidis
35
(SE) or S. Infantis (SINF) with cellular fibronectin (FN) laminin (LN) or MatrigelTM given as the mean
36
value ( standard deviation) of the fold change in comparison to the controls. Asterisks indicate
37
significant differences between the control group and the co-cultivated groups (*: P 0.05).
38
Accepted Manuscript
Figure 4
1
Variations of virulence gene expression of S. Enteritidis (SE) and S. Infantis (SINF) after co-
2
cultivation of bacteria and cellular fibronectin (A), MatrigelTM (B) or laminin (C). Results, given in
3
log10 median of the signal ratio of matrix protein and the control, are referred to 13 out of the 46 genes
4
tested (Table 2) that underwent expression variations. Asterisks indicate significant differences
5
between the control group and the co-cultivated groups (*: P 0.05; **: P 0.01).
6
Accepted Manuscript
Table 1
1
Antibodies used for immunohistochemical investigation 2
antigen clone dilution Source/reference
Laminin (all variants)
rabbit polyclonal L9393
LM: 1:500 FM: 1:50
Sigma-Aldrich, Taufkirchen, Germany
Laminin-332 rabbit polyclonal R14
LM: 1:20000 Prof. M. Aumailly, Cologne, Germany Aumailley and Rousselle, 1999 Fibronectin
(all variants)
rabbit polyclonal (A0245)
LM: 1:2000 FM: 1:200
DakoCytomation, Hamburg, Germany
Fibronectin ED-A domain
IST-9 (IgG1)
LM: 1:600 FM: 1:60
Prof. L. Zardi / Dr. L. Borsi Genova, Italy
Borsi et al., 1987; Carnemolla et al. 1987 Tenascin-C
(all variants)
rabbit polyclonal AB19013
LM: 1:1000 FM: 1:100
Chemicon Int., Temecula, USA
Salmonella common antigen (LPS)
100/353.2 (IgG2a)
FM: 1:100 Chemicon Int., Temecula, USA
LM = immunohistochemistry for light microscopy using the Dako REALTM Detection System
3
FM = fluorescence immunohistochemistry
4
Accepted Manuscript
Table 2
1
Salmonella virulence genes used in microarray expression analysis.
2 3 4
Gen Product Sanger-Classification
16S ribosomal DNA
dnaN DNA polymerase III subunit beta 3.A.7 DNA replication,
restriction/modification, recombination and repair
rpoD RNA polymerase sigma factor 2 Broad regulatory functions
avrA putative inner membrane protein SPI-1
hilC invasion regulatory protein SPI-1
prgK needle complex inner membrane lipoprotein SPI-1
prgJ needle complex minor subunit SPI-1
hilD invasion protein regulatory protein SPI-1
hilA invasion protein transcriptional activator SPI-1
sipA secreted effector protein SPI-1
sipB translocation machinery component SPI-1
sicA secretion chaperone SPI-1
invE cell invasion protein SPI-1
invF invasion regulatory protein SPI-1
ssaT type III secretion system apparatus protein SPI-2
ssaN type III secretion system ATPase SPI-2
ssaV type III secretion system apparatus protein SPI-2
sseG secreted effector protein SPI-2
sseA secretion system chaperone protein SPI-2
ssaB secreted effector protein SPI-2
ssrA sensor kinase SPI-2
ttrR response regulator SPI-2
mgtB Mg2+ transporter SPI-3
misL putative autotransporter SPI-3
Accepted Manuscript
rmbA putative cytoplasmic protein SPI-3
STM4257 putative exported protein SPI-4
STM4259 putative type-I secretion protein SPI-4
pipC cell invasion protein SPI-5
sopB secreted effector protein SPI-5
pipD putative secreted peptidase SPI-5
phoP response regulator in two-component regulatory system with PhoQ
2 Broad regulatory functions
rpoS sigma S (sigma 38) factor of RNA polymerase 2 Broad regulatory functions
relA (p)ppGpp synthetase I 2 Broad regulatory functions
spoT (p)ppGpp synthetase II / guanosine-3',5'-bis pyrophosphate 3'-pyrophosphohydrolase
2 Broad regulatory functions
lon DNA-binding protein 2 Broad regulatory functions
sodC1 copper/zinc superoxide dismutase 4.G Detoxification sodC2 copper/zinc superoxide dismutase 4.G Detoxification
galE UDP-galactose 4-epimerase 1.A.1 Degradation of carbon compounds
fliC flagellar biosynthesis; flagellin 3.C.3 Surface structures
yliH putative cytoplasmic protein 5.H.a Hypothetical protein
clpP proteolytic subunit of clpA-clpP ATP-dependent serine protease
3.B.3 Degradation of proteins, peptides and glycopeptides
htrA
periplasmic serine protease Do, heat shock protein
3.B.3 Degradation of proteins, peptides and glycopeptides
yciG putative cytoplasmic protein 5.H.a Hypothetical protein
adhP alcohol dehydrogenase 1.B.7.b Anaerobic Respiration
osmC putative envelope protein 5.F Adaptions and atypical conditions
pspA phage shock protein 5.F Adaptions and atypical conditions
sopA secreted effector protein 4.I Pathogenicity
sopE2 type III-secreted effector protein 4.I Pathogenicity
spvB hydrophilic protein Plasmide (pSLT)
1
Accepted Manuscript
control SE SINF
A B C
D E F
G H I
K L M
E L
LP
M L E LP
M
E L
LP M
E L
LP M
E
L LP
M E
L
LP M
E L
M LP E
L
LP
M
E
L LP
M
E L
M LP
L E
LP
M
L E LP
M
SM SM
SM
SM
SM SM
SM
SM
SM
SM SM
Tn- C EDA + Fn Ln tFn
Berndt et al. Figure 1
Figure 1
Accepted Manuscript
A B C
D E F
G H I
K L M
L
L E
LP
E
LP
E
LP L L
E LP
L
E
LP
L
E
LP E
L E
LP E
L LP
E
LP SM
E L LP
M
E L
LP M
L E
LP
M
L SM
LM
Berndt et al. Figure 2
Figure 2
Accepted Manuscript
Figure 3 Berndt et al.
0 2 4 6 8 10 12 14 16 18 20
FN LN Matrigel
fol d c h ange
SE SINF
*
*
*
B
0 10 20 30 40 50 60 70 80 90 100
FN LN Matrigel
% adherent bacteria
SE SINF
* A
b
*
*
a
c
Figure 3
