Association of Bordetella dermonecrotic toxin with the extracellular matrix.

Abstract

Bordetella dermonecrotic toxin (DNT) causes the turbinate atrophy in swine atrophic rhinitis, caused by a Bordetella bronchiseptica infection of pigs, by inhibiting osteoblastic differentiation. The toxin is not actively secreted from the bacteria, and is presumed to be present in only small amounts in infected areas. How such small amounts can affect target tissues is unknown.
Fluorescence microscopy revealed that DNT associated with a fibrillar structure developed on cultured cells. A cellular component cross-linked with DNT conjugated with a cross-linker was identified as fibronectin by mass spectrometry. Colocalization of the fibronectin network on the cells with DNT was also observed by fluorescence microscope. Several lines of evidence suggested that DNT interacts with fibronectin not directly, but through another cellular component that remains to be identified. The colocalization was observed in not only DNT-sensitive cells but also insensitive cells, indicating that the fibronectin network neither serves as a receptor for the toxin nor is involved in the intoxicating procedures. The fibronectin network-associated toxin was easily liberated when the concentration of toxin in the local environment decreased, and was still active.
Components in the extracellular matrix are known to regulate activities of various growth factors by binding and liberating them in response to alterations in the extracellular environment. Similarly, the fibronectin-based extracellular matrix may function as a temporary storage system for DNT, enabling small amounts of the toxin to efficiently affect target tissues or cells.

Full text

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RESEARCH ARTICLEOpen Access
Association of Bordetella dermonecrotic toxin
with the extracellular matrix
Aya Fukui-Miyazaki1*, Shigeki Kamitani1, Masami Miyake2, Yasuhiko Horiguchi1
Abstract
Background: Bordetella dermonecrotic toxin (DNT) causes the turbinate atrophy in swine atrophic rhinitis, caused
by a Bordetella bronchiseptica infection of pigs, by inhibiting osteoblastic differentiation. The toxin is not actively
secreted from the bacteria, and is presumed to be present in only small amounts in infected areas. How such small
amounts can affect target tissues is unknown.
Results: Fluorescence microscopy revealed that DNT associated with a fibrillar structure developed on cultured
cells. A cellular component cross-linked with DNT conjugated with a cross-linker was identified as fibronectin by
mass spectrometry. Colocalization of the fibronectin network on the cells with DNT was also observed by
fluorescence microscope. Several lines of evidence suggested that DNT interacts with fibronectin not directly, but
through another cellular component that remains to be identified. The colocalization was observed in not only
DNT-sensitive cells but also insensitive cells, indicating that the fibronectin network neither serves as a receptor for
the toxin nor is involved in the intoxicating procedures. The fibronectin network-associated toxin was easily
liberated when the concentration of toxin in the local environment decreased, and was still active.
Conclusions: Components in the extracellular matrix are known to regulate activities of various growth factors by
binding and liberating them in response to alterations in the extracellular environment. Similarly, the fibronectin-
based extracellular matrix may function as a temporary storage system for DNT, enabling small amounts of the
toxin to efficiently affect target tissues or cells.
Background
Pathogenic bacteria of the genus Bordetella produce
dermonecrotic toxin (DNT), which activates Rho
GTPases through its transglutaminase activity resulting
in deamidation or polyamination [1-3]. DNT is a single
chain polypeptide of 1,464 amino acids, with an
N-terminal region of at least 54 amino acids responsible
for binding to a receptor on target cells [4] and a
C-terminal region of about 300 amino acids conferring
the transglutaminase activity [5]. The receptor for DNT
is still unknown. The activated Rho GTPases cause
aberrant Rho-dependent phenotypes [6,7], which likely
lead to some of the pathological changes observed dur-
ing Bordetella infections. For example, the turbinate
atrophy in atrophic rhinitis, a Bordetella infection of
pigs, is caused by DNT acting on osteoblastic cells
[8-13]. However, there has been no evidence that
DNT is actively secreted from the bacteria, and less
than 0.75% (0.60 ng/109CFU) of produced DNT was
detected in culture supernatant of B. bronchiseptica and
B. pertussis (unpublished data). It is unknown how this
small amount of DNT exerts toxicity against target
cells such as osteoblasts covered by epithelial cells and
connective tissue.
While attempting to identify the receptor for DNT, we
found that DNT associated temporarily with fibronectin
(FN)-based extracellular matrix (ECM), on both DNT-
sensitive and insensitive cells, indicating that the FN
network does not serve as a functional receptor for
DNT. We hypothesized that the FN network functions
as a temporary storage system for DNT, enabling the
small amount of the toxin to effectively reach target
cells across the epithelia and connective tissue.
* Correspondence: fuku-a@biken.osaka-u.ac.jp
1Department of Molecular Bacteriology, Research Institute for Microbial
Diseases, Osaka University, Osaka, Japan
Full list of author information is available at the end of the article
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© 2010 Fukui-Miyazaki et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
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Results
DNT binds to the FN-based ECM network
While attempting to identify a receptor for DNT, we
found that DNT was distributed along with a fibrillar
structure on the surface of MC3T3-E1 cells (Fig. 1A),
suggesting an affinity for some component of the ECM.
This affinity appeared to be dependent on pH: most of
the bound toxin was easily washed away from the cell
surface at pH 7 or 9, whereas a detectable amount of
DNT remained bound after washing at pH 5 (Fig. 1B).
To identify the cellular component associated with
DNT, we tried to cross-link the toxin with its counter-
part using Sulfo-SBED, a trifunctional cross-linker,
which labels the cross-linked molecule with biotin.
Sulfo-SBED-labeled DNT (SBED-DNT), which had a
similar distribution to the native toxin (Fig. 1A-d),
transferred biotin to at least three distinct cellular
components in NP-40 insoluble fraction detected by
Western blotting (Fig. 1C). Only the component with
the highest molecular weight could be isolated by
anion-exchange chromatography (Fig. 1D and 1E), and
identified as mouse FN by mass spectrometry. FN is a
major component organizing the ECM. We examined if
the toxin colocalizes with the FN network by staining
FN or other ECM components, such as collagen type I
and laminin. DNT was found to be well colocalized with
the FN network and partly colocalized with the collagen
type I, but not colocalized with laminin (Fig. 2).
Besides MC3T3-E1 cells, which are sensitive to DNT,
DNT-insensitive Balb3T3 cells also showed the colocali-
zation of DNT with the FN network (Fig. 3). FN-null
cells, which are fibroblastic cells isolated from FN
Figure 1 DNT is associated with the fibrillar structure on MC3T3-E1 cells. (A) The cells were treated with DNT (a and b), 5-FAM-DNT (c), or
SBED-DNT (d) as mentioned in Methods. The cells were stained without wash as follows. DNT was detected with a combination of anti-DNT
polyclonal antibody and Alexa 488-conjugated secondary antibody (b). The DNT-treated cells were stained with only the secondary antibody for
the control (a). 5-FAM-DNT was visualized with direct fluorescence microscopy (c). SBED-DNT was detected with Alexa 488-conjugated
streptavidin (d). Note that the association of DNT with the fibrillar structure was observed independently of the detection method. Bar, 5 μm.
(B) MC3T3-E1 cells were incubated with DNT at different pH and stained with anti-DNT polyclonal antibody. The cells were washed once (lower
panels) or not washed (upper panels) before fixation. Bar, 5 μm. (C) Cellular components cross-linked by SBED-DNT. MC3T3-E1 cells were
incubated with (lane 2) or without (lane 1) SBED-DNT. After the cross-linking procedure, the insoluble fraction was prepared as described in
Methods and subjected to SDS-PAGE with a 6% acrylamide gel containing 6 M urea under reducing conditions. Cellular components labeled by
biotin through SBED were detected by Western blotting with HRP-conjugated streptavidin. Arrows indicate cellular components cross-linked with
SBED-DNT. (D) Mini Q column chromatographic profile of the insoluble fraction of MC3T3-E1 cells treated and cross-linked with SBED-DNT. The
cellular component with the higher molecular weight was eluted in fractions 6 to 8 (bold bar). (E) SDS-PAGE of fraction 7. The cellular
component with the higher molecular weight is indicated with an asterisk.
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knock-out mouse embryos and is sensitive to DNT, did
not show a fibrillar distribution of the toxin (Fig. 3).
These result indicate that the association of DNT with
FN is not related to the intoxication. When human FN
was supplied to the culture, FN-null cells showed the
colocalization of the toxin and FN. In contrast, DNT
did not colocalize with the FN network developed on
MRC-5 cells (Fig. 3). These results suggest that DNT
does not interact directly with FN, and another cellular
component, which is present in the culture of FN-null
cells but not MRC-5 cells, is necessary for the interac-
tion. In fact, MRC-5 cells supplemented with the culture
supernatant of FN-null cells showed the colocalization
of DNT and the FN network (Fig. 4). Treatment with
heat at 95°C or proteinase K abolished the ability of the
culture supernatant to recruit DNT to the FN network,
which indicates that the unknown component exists in
the culture supernatant of FN-null cells and contains a
protein moiety (data not shown).
Screening for a molecule mediating the colocalization of
DNT and the FN network
We tried to isolate the unknown component from the
culture supernatant of FN-null cells by ion-exchange
chromatography (Fig. 5A). Each fraction eluted by Mono
Q anion-exchange chromatography was added to the
culture of MRC-5 cells, and checked for the ability to
recruit DNT to the FN network. Simultaneously, each
fraction was subjected to SDS-PAGE and proteins in the
fractions were identified by mass spectrometry. Fraction
4 apparently induced the association of DNT with the FN
network on MRC-5 cells (Fig. 5B). Mass spectrometry
revealed that fraction 4 contains ECM-related proteins
such as nidogen-2 in an N-terminally truncated form
(open arrowhead), and lysyl oxidase-homolog 2 (LOXL2)
and 3 (LOXL3) (Fig. 5C). Similar results were obtained
from the culture supernatant of MC3T3-E1 cells: the
truncated form of nidogen-2 (open arrowhead) and
LOXL3 were found in fraction 4, which induced the asso-
ciation of DNT with the FN network on MRC-5 cells
(Fig. 5D). LOXL2 was expressed at neither the mRNA
nor protein level in MC3T3-E1 cells, which show inten-
sive colocalization of DNT and the FN network (Fig. 3).
LOXL3 supplemented to the culture did not induce the
colocalization of DNT with the FN network on MRC-5
cell (data not shown). Therefore, the truncated form of
nidogen-2 but not LOXL2 or 3 is considered to be a pos-
sible candidate for the unknown intermediate between
Figure 2 Colocalization of DNT with the ECM components. MC3T3-E1 cells incubated with DNT were stained with anti-DNT monoclonal
antibody or polyclonal antibody against FN, collagen type I or laminin. Bars, 5 μm.
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DNT and FN. Nidogen-2 was found to organize a net-
work on the cells and colocalize with DNT and FN
(Fig. 6).
DNT is liberated from the FN network and affects
sensitive cells
We examined whether DNT liberated from the FN net-
work was still active (Fig. 7). FN-null cells supplemented
with or without human FN were treated with DNT, and
the amount of toxin that diffused from the cells after
replacement of the medium was measured by ELISA.
DNT gradually diffused from the FN-supplemented FN-
null cells in 60 min (Fig. 7A). Its concentration was
about three times that which diffused from unsupple-
mented cells. The diffused toxin caused the reorganiza-
tion of actin stress fibers in MC3T3-E1 cells, indicating
that it was still active even after its association with, and
liberation from, the FN network (Fig. 7B).
Discussion
Here, we found that DNT temporarily associated with the
FN network on cells. FN, a major component of the ECM,
is mainly produced by fibroblasts and organized into a
fibrillar network through binding to cell surface receptors,
integrins[14-16].A DNT mutant deficientin
transglutaminase activity was also associated with the FN
network (data not shown), indicating that the enzymatic
activity of DNT is not required for the association.
Because deletion mutants of DNT, in which any of the
regions is missing, and heat-inactivated DNT did not asso-
ciated with the FN network (data not shown), the overall
structure of the toxin may be crucial to the association.
DNT did not colocalize with the FN network generated by
MRC-5 cells, suggesting that it interacts with FN not
directly, but via another cellular component. Nidogen-2 in
an N-terminally truncated could be a candidate for the
component, because it was present in only the fraction
which induced the association of DNT with the FN net-
work on MRC-5 cells, whereas full-length nidogen-2 did
not. Although its biological importance is not fully under-
stood, nidogen-2 is known to interact with various mole-
cules in the ECM [17]. The nature of the truncated
nidogen-2 is currently unknown. How the truncated nido-
gen-2 mediates the association between DNT and the FN
network is not known either. At least, we observed that
nidogen-2 was colocalized with not only FN but also DNT
in the fibrillar structure. SBED-DNT crosslinked to two
distinct components in addition to FN (Fig. 1C). These
two components might be other candidates to intermedi-
ate the association between DNT and the FN network.
Figure 3 Colocalization of DNT with the FN network on various cells. Cells were incubated with DNT and stained with anti-DNT
monoclonal antibody and anti-FN polyclonal antibody. FN-null cells were incubated with or without human FN (hFN) before DNT treatment.
Bars, 5 μm.
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However, they could not be isolated by combinations of
anion- and cation-exchange chromatographies, probably
because of their instability. In addition, the living cells,
some cell membrane proteins, and/or the fibrillar struc-
ture of FN may be also required, because we could not
reproduce the association of DNT with FN in the presence
of the culture supernatant of FN-null cells by in vitro tech-
niques such as ELISA and immunoprecipitation (data not
shown). DNT may associate with the FN network by a
complicated mechanism involving the truncated nidogen-
2 and other cellular components. We are now conducting
further work to elucidate this issue.
The association of DNT with the FN network was
seen in not only DNT-sensitive cells but also insensitive
cells, which indicates that the FN network neither serves
as a receptor for the toxin nor is involved in the intoxi-
cating procedures of the toxin on sensitive cells. Instead,
the FN network may serve as a transient storage site for
DNT, allowing the toxin to reach target cells or tissues
more efficiently. Components of the ECM including FN
are known to bind and regulate various growth factors
such as insulin-like growth factor (IGF), fibroblast
growth factor (FGF), transforming growth factor-beta
(TGF-b), hepatocyte growth factor (HGF), and vascular
endothelial growth factor (VEGF) [18,19]. These growth
factors are released from the ECM in response to altera-
tions in the extracellular environment and exert biologi-
cal effects to regulate cell survival, proliferation, and
differentiation. For example, VEGF is associated with
the ECM via FN or heparan sulfate at acidic pH. When
the pH of the extracellular milieu increases, VEGF is
released from the ECM network and activates its func-
tional receptor to induce angiogenesis [20,21]. This
pH-dependent association of VEGF is considered a key
mechanism determining the direction of newly devel-
oped blood vessels in wound healing and tumor metas-
tasis. The association of DNT with the FN network was
also dependent on the pH of the extracellular environ-
ment. Bordetella infections are reported to be accompa-
nied by necrosis or the desquamation of superficial
epithelial layers with inflammatory responses [22,23].
These events may facilitate the exposure of newly gener-
ated ECM containing FN. The inflammatory locus is
reportedly characterized by local acidosis due to lactic
acid production [24]. FN is actively produced by fibro-
blasts and osteoblasts, mesenchymal cells, which could
be targets for DNT. Therefore, it is conceivable that
DNT binds to the ECM containing FN at low pH
in inflammatory areas during an infection, and by
repeatedly associating with and diffusing from the FN
network, moves deep into tissue where the density of
FN should be higher, eventually reaching target cells.
This may explain how DNT, which is not secreted by
bacteria and is present at low concentrations in extra-
bacterial milieus, can affect target tissues in Bordetella
infections such as atrophic rhinitis.
Conclusions
DNT associates temporarily with FN-based ECM network.
The association seems to be mediated by the truncated-
form of nidogen-2 and/or some cellular components,
which have an affinity to the FN network. It is likely that
the FN network does not function as a specific receptor
but serves as a temporary storage system for DNT,
enabling the small amount of the toxin to effectively reach
target cells across the epithelia and connective tissue.
Methods
Cell culture
Mouse preosteoblastic cells MC3T3-E1 were cultured in
alpha modified Eagle’s medium (a-MEM) supplemented
Figure 4 Colocalization of DNT with the FN network on MRC-5
cells supplemented with the culture supernatant of FN-null
cells. MRC-5 cells, which were pre-cultured with or without the
culture supernatant of FN-null cells (FN-null CS), were incubated
with DNT and stained with anti-DNT monoclonal antibody and anti-
FN polyclonal antibody. Bars, 5 μm.
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with 10% fetal calf serum (FCS). Mouse embryonic
fibroblasts Balb3T3 and human fibroblasts MRC-5 were
cultured in Dulbecco’s modified Eagle’s medium
(DMEM) supplemented with 10% FCS. FN-null cells,
which were kindly provided by Dr. Sottile [25], were
maintained in an 1:1 mixture of Cellgro (Mediatech)
and Aim V (GIBCO/Invitrogen).
Reagents and antibodies
Human plasma FN was purchased from Sigma. Collagen
to coat culture plates and slide glasses was from Nitta
gelatin Co. (Cellmatrix, Osaka, Japan). The monoclonal
(FN-15, F7387) and polyclonal (F3648) antibodies against
FN and polyclonal antibody against laminin (L9393) were
obtained from Sigma. The anti-mouse nidogen-2 (M-300,
sc-33143) and anti-collagen type I (234168) antibodies
were from Santa Cruz and Calbiochem, respectively. The
anti-DNT monoclonal antibody 2B3 and anti-DNT poly-
clonal antibody were prepared as reported [4,26]. Alexa
488-conjugated goat anti-rabbit IgG, Alexa 546-conjugated
goat anti-mouse IgG, and Alexa 488-conjugated streptavi-
din were from Molecular Probes/Invitrogen. Horseradish
peroxidase (HRP)-conjugated streptavidin was from
Chemicon. DNT that is N-terminally fused with hexahisti-
dine was obtained as reported [27].
Sulfo-SBED, a trifunctional cross-linking reagent, was
purchased from Thermo scientific. 5-carboxyfluorescein,
succinimidyl ester (5-FAM, SE) was obtained from
Molecular Probes/Invitrogen. For conjugation, DNT was
dialyzed against 0.1 M NaHCO3, pH 8.3, mixed with
Figure 5 Screening for a molecule mediating the association of DNT with the FN network. (A) Profile of Mono Q anion-exchange
chromatography of the culture supernatant of FN-null cells. (B) The association of DNT with the FN network of MRC-5 cells supplemented with
each fraction from the chromatography. MRC-5 cells seeded in a 24-well plate were incubated overnight with eluted fractions. The next day, the
cells were treated with 2 μg/ml of DNT, and stained with anti-DNT polyclonal antibody as described in Methods. Bar, 5 μm. (C) Each fraction
from the chromatography was subjected to SDS-PAGE followed by silver staining. The arrows and arrowheads indicate the proteins identified by
mass spectrometry. The asterisk indicates contaminated human keratin. (D) The fractions from chromatography with the culture supernatant of
MC3T3-E1 cells. Nidogen-2 was detected at approximately 200 kDa, and the smaller variants of nidogen-2 are presumed to be N-terminally
truncated, based on the results of mass spectrometry (arrowheads). Note that the band indicated by open arrowhead is present in fraction 4
inducing the association of DNT with the FN network.
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Sulfo-SBED or 5-FAM, SE at a molar ratio of 1:32, and
incubated at room temperature for 30 min. After
incubation, the unconjugated reagent was removed by
gel filtration with a PD-10 column (GE Healthcare).
Immunofluorescent staining of DNT-treated cells
MC3T3-E1, Balb3T3, and MRC-5 cells were seeded at
50,000 cells/cm2in wells of a 24-well plate with cover
glasses and grown overnight. FN-null cells were cultured
overnight on collagen-coated cover glasses in Cellgro-
Aim V with or without 10 μg/ml of human FN. The
next day, the medium was replaced with a fresh batch
containing 2 μg/ml of DNT, 5-FAM-conjugated DNT
(5-FAM-DNT) or SBED-conjugated DNT (SBED-DNT),
and the cells were incubated for 15 min at 37°C. The
cells were then fixed with 3% paraformaldehyde in Dul-
becco’s modified phosphate-buffered saline (D-PBS (-))
for 10 min and treated with primary antibodies for 1 h,
and subsequently secondary antibodies for 30 min in the
presence of 10% FCS. The cells were washed three times
with D-PBS (-) after each procedure. The cells were
mounted in Fluoromount (Diagnostic BioSystems) and
imaged with an OLYMPUS BX50 microscope controlled
by SlideBook 4.0 (Intelligent Imaging Innovation, Inc.).
Anti-DNT polyclonal or monoclonal antibodies were
used at 10 μg/ml for DNT staining. FN, collagen typeI,
laminin, and nidogen-2 were stained with the respective
antibodies at concentrations indicated in the instruction
manuals.
Cross-linking of MC3T3-E1 cells with SBED-conjugated
DNT
Confluent MC3T3-E1 cells in a 10-cm dish were treated
with 2.5 μg/ml of SBED-DNT at 37°C for 15 min and
then exposed to UV light at 365 nm for 5 min. The cells
were washed with D-PBS (-) twice and solubilized with
D-PBS (-) containing 1% NP-40 and 1% protease inhibi-
tor cocktail (Nacalai, Kyoto, Japan) at 4°C for 60 min.
The solubilized cells were centrifuged at 10,000 × g for
20 min at 4°C and the supernatant and pellet were
collected as the soluble fraction and insoluble fraction,
respectively. The insoluble fraction was sonicated in
D-PBS (-) containing 5 μg/ml of DNase I and 8 M urea.
After centrifugation, the supernatant was injected into a
Mini Q column (0.32 × 3 cm, GE Healthcare), and eluted
with a gradient of 0-1 M NaCl in 20 mM Tris-HCl
(pH 8.5), containing 8 M urea, using the SMART system
(GE Healthcare).
Screening for components intermediating the association
between DNT and the FN network
FN-null cells or MC3T3-E1 cells were cultured in FCS-
free DMEM or a-MEM for 72 h. The supernatant of
the culture was dialyzed against 20 mM Tris-HCl,
pH 8.5 containing 0.5 M NaCl, and subjected to anion-
exchange chromatography with a HiTrap Q column
(0.7 × 2.5 cm, GE Healthcare). The materials absorbed
to the column were eluted in 1-ml fractions with a lin-
ear gradient of 0.5-1 M NaCl, and each fraction was
tested for the ability to recruit DNT to the fibrillar
structure on MRC-5 cells using immunofluorescence
microscopy. The positive fractions were collected,
appropriately diluted, and mixed with 5% CHAPS and
10 M urea to make a solution of 20 mM Tris-HCl, pH
8.5, containing 50 mM NaCl, 0.5% CHAPS and 6 M
urea. The sample was subjected to Mono Q anion-
exchange chromatography, and eluted with a linear gra-
dient of 0.05-1 M NaCl. The eluted fractions were
examined again for the ability to recruit DNT to the
fibrillar structure on MRC-5 cells. Proteins contained in
the positive fraction were identified by mass spectrome-
try as mentioned below.
DNT diffusion assay
FN-null cells were seeded in wells of a 24-well plate at
25,000 cells/cm2and grown overnight. The next day,
Figure 6 Colocalization of nidogen-2 and DNT or FN. MC3T3-E1
cells incubated with DNT were stained with anti-nidogen-2
polyclonal antibody, and anti-DNT or anti-FN monoclonal
antibodies. Bars, 5 μm.
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the cells were washed well with Cellgro-Aim V and
incubated overnight in the same medium with or with-
out 10 μg/ml of human FN. The culture medium was
replaced with a fresh batch containing 2.5 μg/ml of
DNT and the cells were incubated for 15 min at 37°C.
After three washes with FCS-free DMEM, the cells were
further incubated in the fresh medium. The culture
supernatant was taken at the indicated time point, and
an aliquot was applied to MC3T3-E1 cells without dilu-
tion. After incubation at 37°C overnight, the cells were
examined for actin stress fibers as described previously
[27]. Another aliquot of the culture supernatant was
examined for DNT by sandwich-ELISA, performed with
a 96-well plate coated with anti-DNT polyclonal anti-
body. After blocking with 0.2% BSA at 4°C overnight,
each sample was added to the plate in triplicate and
incubated for 2 h at 37°C. The plate was treated with
biotin-labeled anti-DNT antibody, followed by HRP-
conjugated streptavidin for 1 h at 37°C. BM Blue POD
substrate (Roche) was used as an HRP substrate and the
reaction was stopped by 1 M H2SO4. The wells were
washed four times with D-PBS (-) containing 0.05%
Tween-20 between each step. The concentration of
DNT was estimated from a standard curve made with a
DNT preparation.
Other methods
Protein concentrations were determined using BCA Pro-
tein Assay Reagents (Thermo Scientific). For the mass
spectrometric analysis, samples were subjected to SDS-
PAGE, followed by staining using a Silver stain MS kit
(Wako). Visualized proteins were exercised from the gels,
and digested with trypsin according to a method
described elsewhere [28]. Mass spectrometric data were
analyzed with the MASCOT program (Matrix Science
Ltd.). The statistical differences among groups of data
were analyzed by one-way analysis of variance (ANOVA),
followed by a Bonferroni posttest, using GraphPad Prism
software version 4 (GraphPad Software, Inc.)
Abbreviations
(DNT): Dermonecrotic toxin; (ECM): extracellular matrix; (FN): fibronectin
Acknowledgements
We are grateful to Dr. Sottile (University of Rochester Medical Center, NY,
USA) for providing the FN-null cells. This work was supported in part by
Grants-in-Aid for Scientific Research from the Ministry of Education, Culture,
Science, and Technology of Japan.
Author details
1Department of Molecular Bacteriology, Research Institute for Microbial
Diseases, Osaka University, Osaka, Japan.2Laboratory of Veterinary Public
Health, Department of Veterinary Environmental Sciences, Osaka Prefecture
University, Osaka, Japan.
Figure 7 DNT associated with the FN network diffuses from the cell surface and affects sensitive cells. (A) The concentration of DNT
diffused from FN-null cells supplemented with hFN (open triangles) or not (closed triangles). The culture supernatant of the cells was obtained
as described in Methods, and the DNT concentration was determined. As a control, the medium incubated without FN-null cells (closed squares)
was prepared in the same manner. The abscissa indicates the time after the washing of DNT-treated cells. Each plot represents the mean ± S.D.
(n = 3). Asterisks indicate significant differences (P < 0.001). (B) Stress fiber-inducing activity of DNT liberated into the culture supernatant. The
culture supernatant was obtained 15 min after washing in the experiments of panel A, and examined for DNT activity on MC3T3-E1 cells. Actin
fibers were visualized by rhodamine-phalloidin. The left panels show MC3T3-E1 cells incubated with each culture supernatant and the right
panels show the cells incubated with DNT. The experiments were performed three times and representative results are shown. Bar, 5 μm.
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Authors’ contributions
AF-M participated in the design and performed all experiments and drafted
the manuscript. SK and MM contributed to the interpretation of data. YH
obtained funding for designed the research and critically revised the
manuscript. All authors read and approved the final manuscript.
Received: 20 May 2010 Accepted: 25 September 2010
Published: 25 September 2010
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doi:10.1186/1471-2180-10-247
Cite this article as: Fukui-Miyazaki et al.: Association of Bordetella
dermonecrotic toxin with the extracellular matrix. BMC Microbiology 2010
10:247.
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