Group Title: BMC Microbiology
Title: Gene expression profile and pathogenicity of biofilm-forming Prevotella intermedia strain 17
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Title: Gene expression profile and pathogenicity of biofilm-forming Prevotella intermedia strain 17
Physical Description: Book
Language: English
Creator: Yamanaka, Takeshi
Furukawa, Tomoyo
Matsumoto-Mashimo, Chiho
Yamane, Kazuyoshi
Sugimori, Chieko
Nambu, Takayuki
Mori, Naoki
Nishikawa, Hiroyuki
Walker, Clay
Leung, Kai-Poon
Fukushima, Hisanori
Publisher: BMC Microbiology
Publication Date: 2009
 Notes
Abstract: BACKGROUND:Prevotella intermedia (P. intermedia), a gram-negative, black-pigmented anaerobic rod, has been implicated in the development of chronic oral infection. P. intermedia strain 17 was isolated from a chronic periodontitis lesion in our laboratory and described as a viscous material producing strain. The stock cultures of this strain still maintain the ability to produce large amounts of viscous materials in the spent culture media and form biofilm-like structures. Chemical analyses of this viscous material showed that they were mainly composed of neutral sugars with mannose constituting 83% of the polysaccharides. To examine the biological effect of the extracellular viscous materials, we identified and obtained a naturally-occurring variant strain that lacked the ability to produce viscous materials in vitro from our stock culture collections of strain 17, designated as 17-2. We compared these two strains (strains 17 versus 17-2) in terms of their capacities to form biofilms and to induce abscess formation in mice as an indication of their pathogenicity. Further, gene expression profiles between these two strains in planktonic condition and gene expression patterns of strain 17 in solid and liquid cultures were also compared using microarray assays.RESULTS:Strain 17 induced greater abscess formation in mice as compared to that of the variant. Strain 17, but not 17-2 showed an ability to interfere with the phagocytic activity of human neutrophils. Expression of several genes which including those for heat shock proteins (DnaJ, DnaK, ClpB, GroEL and GroES) were up-regulated two to four-fold with statistical significance in biofilm-forming strain 17 as compared to the variant strain 17-2. Strain 17 in solid culture condition exhibited more than eight-fold up-regulated expression levels of several genes which including those for levanase, extracytoplasmic function-subfamily sigma factor (sE; putative) and polysialic acid transport protein (KpsD), as compared to those of strain 17 in liquid culture media.CONCLUSION:These results demonstrate that the capacity to form biofilm in P. intermedia contribute to their resistance against host innate defence responses.
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Research article

Gene expression profile and pathogenicity of biofilm-forming
Prevotella intermedia strain 17
Takeshi Yamanaka*1, Tomoyo Furukawa', Chiho Matsumoto-Mashimo1,
Kazuyoshi Yamanel, Chieko Sugimoril, Takayuki Nambu1, Naoki Mori1,
Hiroyuki Nishikawal, Clay B Walker2, Kai-Poon Leung3 and
Hisanori Fukushima1


Address: 'Department of Bacteriology, Osaka Dental University, Osaka, Japan, 2Department of Oral Biology, College of Dentistry, University of
Florida, Gainesville, FL 32610-0424, USA and 3Microbiology Branch, U.S. Army Dental and Trauma Research Detachment, Walter Reed Army
Institute of Research, Great Lakes, IL 60088, USA
Email: Takeshi Yamanaka* yamanaka@cc.osaka-dent.ac.jp; Tomoyo Furukawa tomomo-f@ezweb.ne.jp; Chiho Matsumoto-
Mashimo mashimo@cc.osaka-dent.ac.jp; Kazuyoshi Yamane yamane@cc.osaka-dent.ac.jp; Chieko Sugimori chieko@cc.osaka-dent.ac.jp;
Takayuki Nambu nambu-t@cc.osaka-dent.ac.jp; Naoki Mori mori3636@ezweb.ne.jp; Hiroyuki Nishikawa nishikawa.dc@kha.biglobe.ne.jp;
Clay B Walker walkercl@ufl.edu; Kai-Poon Leung Kai.Leung@amedd.army.mil; Hisanori Fukushima hisanori@cc.osaka-dent.ac.jp
* Corresponding author


Published: 16 January 2009
8MC Microbiology 2009, 9:11 doi:10.1186/1471-2180-9- I
This article is available from: http://www.biomedcentral.com/1471-2180/9/1 I


Received: 29 August 2008
Accepted: 16 January 2009


2009 Yamanaka 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 reproduction in any medium, provided the original work is properly cited.



Abstract
Background: Prevotella intermedia (P. intermedia), a gram-negative, black-pigmented anaerobic rod, has been
implicated in the development of chronic oral infection. P. intermedia strain 17 was isolated from a chronic
periodontitis lesion in our laboratory and described as a viscous material producing strain. The stock cultures of
this strain still maintain the ability to produce large amounts of viscous materials in the spent culture media and
form biofilm-like structures. Chemical analyses of this viscous material showed that they were mainly composed
of neutral sugars with mannose constituting 83% of the polysaccharides. To examine the biological effect of the
extracellular viscous materials, we identified and obtained a naturally-occurring variant strain that lacked the
ability to produce viscous materials in vitro from our stock culture collections of strain 17, designated as 17-2. We
compared these two strains (strains 17 versus 17-2) in terms of their capacities to form biofilms and to induce
abscess formation in mice as an indication of their pathogenicity. Further, gene expression profiles between these
two strains in planktonic condition and gene expression patterns of strain 17 in solid and liquid cultures were also
compared using microarray assays.
Results: Strain 17 induced greater abscess formation in mice as compared to that of the variant. Strain 17, but
not 17-2 showed an ability to interfere with the phagocytic activity of human neutrophils. Expression of several
genes which including those for heat shock proteins (Dnaj, DnaK, CIpB, GroEL and GroES) were up-regulated
two to four-fold with statistical significance in biofilm-forming strain 17 as compared to the variant strain 17-2.
Strain 17 in solid culture condition exhibited more than eight-fold up-regulated expression levels of several genes
which including those for levanase, extracytoplasmic function-subfamily sigma factor (GE; putative) and polysialic
acid transport protein (KpsD), as compared to those of strain 17 in liquid culture media.
Conclusion: These results demonstrate that the capacity to form biofilm in P. intermedia contribute to their
resistance against host innate defence responses.


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Background
Prevotella intermedia, a gram-negative, black-pigmented
anaerobic rod, is frequently isolated from periodontal
pockets of patients with chronic periodontitis [1], acute
necrotizing ulcerative gingivitis [2], pregnancy gingivitis
[3], and endodontic lesions [4-6]. This organism pos-
sesses a number of virulent factors that underlie it's path-
ogenic potential for causing infections [7-11].

P. intermedia strain 17 was initially isolated from a chronic
periodontitis lesion in our laboratory [12] and some of its
phenotypic characteristics were determined. Among these
included the ability of the organism to: (a) produce vis-
cous materials in vitro [12]; (b) invade human oral epithe-
lial cells [13]; and (c) stimulate CD4+ T cells expressing
Vp8, Vpl2 and Vl17 [14]. More recently, the whole
genome sequence of strain 17 was determined by The
Institute for Genomic Research (TIGR; Rockville, MD,
USA) [151.

In our earlier study, we demonstrated that a clinical iso-
late of Prevotella nigrescens is able to produce extracellular
viscous material that might contribute to its biofilm for-
mation [16]. In this context, we hypothesized that the
ability of P. intermedia strain 17 to produce viscous mate-
rials might be essential for its biofilm formation. In this
study, we describe the chemical composition of the vis-
cous materials as determined by means of high perform-
ance liquid chromatography (HPLC) and colorimetry. To
define the role of the extracellular viscous materials in
biofilm formation, we identified and obtained a natu-
rally-occurring variant strain that lacked the ability to pro-
duce viscous materials in vitro from our stock culture
collections of strain 17, designated as 17-2. We compared
the ability of these two strains (strains 17 versus 17-2) in
their ability to form biofilms and to induce abscess forma-
tion in mice as an indication of their pathogenicity. Fur-
ther, we sought to determine the gene expression profiles
associated with the biofilm formation by these two strains
using microarray assays.

Results
Viscosity of spent culture medium
Stock cultures of P. intermedia strain 17 were transferred to
enriched-trypticase soy broth (enriched-TSB) and grown
for 48 h. The viscosities of spent culture media were meas-
ured by a rotary viscometer. All tested P. intermedia strain
17 stocks, with the exception of one particular stock
strain, designated as strain 17-2, produced materials in
vitro that were highly viscous as compared to the control
TSB medium. In contrast, the viscosity of spent culture
medium obtained from strain 17-2 was similar to that of
the control TSB medium (Fig. 1).


2.0 -


1.5 -


1.0'-


Strain 17


Strain 17-2


Figure I
Viscosities of the spent culture media of Prevotella
intermedia strains 17 and 17-2. Viscosities of the spent
culture media obtained from Prevotella intermedia strains 17
and 17-2 were measured by a rotary viscometer. The viscos-
ity of the enriched-TSB medium was measured as a control.
Bars indicate standard deviations.


Cell surface associated structures
SEM observations on cells from colonies of these strains
growing on blood agar plates revealed that strain 17 had
dense meshwork-like structures around the cells (Fig. 2A),
but strain 17-2 lacked this phenotype (Fig. 2B). The lack
of abilities to produce viscous materials in culture
medium and to form meshwork-like structures around
cells on strain 17-2 were stably maintained despite repet-
itive passages in vitro or in animals (data not shown).

Biofilm formation assay
The ability to form biofilm was investigated for strains 17
and 17-2 using crystal violet microtiter plate assay. Strain
17 was consistently able to form biofilm on flat-bottomed
polystyrene microtiter plates, whereas strain 17-2 showed
poorer biofilm formation (Fig. 3A). Quantitative analysis
as measuring the optical density of destined biofilms at
570 nm revealed that the ability of strain 17 to form bio-
film was significantly greater than that of strain 17-2 (p <
0.01) (Fig. 3B).

Morphology and chemical composition of the viscous
materials
Negative staining of the viscous material isolated from
strain 17 culture supernatants revealed that the viscous
material was made up of fine fibrous structures formed in
curly bundles (Fig. 4). Chemical analyses of this purified
material showed that it primarily consisted of neutral sug-
ars and small amounts of uronic acid and amino sugars
(Table 1), with mannose constituting 83% of the polysac-
charide (Table 2).




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Figure 2
Cell surface structures of Prevotella intermedia strains
17 and 17-2. Scanning electron micrographs showing the
surface structures of Prevotella intermedia strains 17 and 17-2.
The specimen was prepared from a colony of each strain
grown on a blood agar plate. Strain 17 had dense meshwork-
like structures surrounding the cell surfaces (A), but strain
17-2 lacked this phenotype (B). Bars = 2 rim.


Gene expression profiles of P. intermedia strains 17 and
17-2
To see what kind of gene expression events induce pheno-
typic differences on P. intermedia, we compared gene
expression patterns between strains 17 and 17-2, the
respective viscous material producing and non-producing
strains using microarray analysis. To determine the appro-
priate time point for isolating total RNA, we first observed
the morphological changes of cell surface structures in
each strain along with the bacterial growth. In general, the
growth of strain 17-2 was faster than that of strain 17,
entering into an exponential phase at around 12 h and
reaching the plateau in 24 h (Fig. 5A, open rhombus).
Strain 17-2 did not show the presence of cell-associated
fibrous materials at any stage of the growth cycle (Fig. 5C).


By contrast, strain 17 showed a slower growth rate (Fig.
5A, hatched square) with a longer exponential growth
phase. Morphological observation of cultures at different
stages of growth revealed that strain 17 exhibited cell sur-
face-associated meshwork-like structures at 12 h and the
structures became denser with time (Fig 5B). From these
preliminary data, 12 h-old cultures of strains 17 and 17-2
were chosen for a comparison of gene expression patterns.
When the microarray expression data for strains 17 and
17-2 were compared, a total of 11 genes were up-regulated
by at least two-fold with statistic significance (p < 0.05) in
biofilm-forming P. intermedia strain 17 (Table 3). The
expression data demonstrated that several heat shock pro-
tein (HSP) genes, such as dnaj, dnaK, groES, groEL and clpB
were up-regulated in strain 17 (Table 3). We also identi-
fied two genes down-regulated at least two-fold in strain
17 (PINA2115: hypothetical protein; PINA2117: sterol-
regulatory element binding protein (SREBP) site 2 pro-
tease family). The original raw data files have been depos-
ited in Center for Information Biology gene Expression
database (CIBEX; Mishima, Japan; CIBEX accession:
CBX27) [171.

mRNA levels for HSPs validated by real-time RT-PCR
In the microarray analysis, we identified that several of the
heat shock protein genes were up-regulated in strain 17 as
compared with those of strain 17-2. The increased expres-
sion levels of these genes were validated in an independ-
ent experiment by real-time RT-PCR using the 16S rRNA
gene as the endogenous control. Annotations of these
genes (PIN0281, PINA1058, PINA1756, PINA1797,
PINA1798, and PINA2006) on TIGR data base were
described in Table 3. Except PIN0281, five out of six of
tested genes showed an at least fivefold increased average
expression levels in strain 17 as confirmed by the quanti-
tative real-time RT-PCR. Although PIN0281 showed
about a three-fold up-regulation in strain 17 by the micro-
array analysis, the average of increased expression level of
PIN0281 was less than two-fold in the real-time RT-PCR
analyses (Fig. 6).

The data obtained from the microarray analysis as well as
the real time RT-PCR showed that several of HSP genes
were up-regulated in strain 17 in 12 h-old cultures as com-
pared with those of strain 17-2. Next, we addressed the
question of whether the different expression levels of HSP
genes between the two strains are due to a lag of growth
because strain 17 showed a slower growth rate than that
of strain 17-2 (Fig. 5). The relative expression levels of
HSP genes through the culture period were obtained using
real time RT-PCR by the strain. In strain 17, the expression
levels of these genes were fluctuating; increased in early
exponential phase (6 h to 12 h), decreased once in the
middle of exponential phase (18 h to 24 h), and then
slightly increased again in early stationary phase. By con-


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A


B


Strain 17

ACJ


Strain 17-2


Exp. 1


-'S


Exp. 2






Exp. 3


Strain 17-2


Figure 3
Biofilm formation on microtiter plates. Biofilm production of Prevotella intermedia strains 17 and 17-2 on polystyrene
microtiter plates: a representative pair of microtitier plate wells from each experiment stained with 0.1 % crystal violet solution
after 24 h of incubation (A). The quantitative analysis of biofilm production as measuring the optical density of destined bio-
films at 570 nm (B). Bars indicate standard deviations.


trast, strain 17-2 did not show such fluctuated transcrip-
tional levels in all HSP genes through the culture period
(Fig. 7). Judging from the comparison between strains 17
and 17-2 at 12 h-old cultures (Fig. 6), strain 17-2 seems to
keep the expression levels of these HSP genes very low.

Abscess induction in mice
To examine the influence of the biofilm phenotype on
pathogenicity of P. intermedia, the abilities of strains 17
Table I: Amount of neutral sugar, uronic acid and amino-sugar
in the viscous material isolated from Prevotella intermedia strain
17


Sugar


Amount (gtg/mg)


Neutral sugar
Uronic acid
Amino-sugar


795.5
28.8


and 17-2 to induce abscesses in mice were compared. An
injection of 500 pil of strain 17 at a concentration of 107
CFU/ml induced abscesses in mice (Fig. 8, left panel). In
contrast, injection of a similar amount of strain 17-2 at the
same growth phase did not induce abscesses in mice. A
much higher cell concentration (109 CFU/ml) of strain

Table 2: Neutral sugar components of the exopolysaccharide
isolated from Prevotella intermedia strain 17


Neutral sugar

Mannose
Glucose
Galactose
Arabinose
Xylose
Rhamnose
Ribose


Amount (gtg/mg)


684.7
53.5
29.2
17.3


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I1~


E
0
r0
L 0.5

0


Strain 17


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Figure 4
Transmission electron microscopy of negatively stained exopolysaccharides isolated from Prevotella intermedia
strains 17 culture supernatants. Note the fine fibrous structures that are formed in bundles. Bar = 500 nm.


17-2 was required to induce abscesses in mice (Fig 8, right
panel). However, an injection of a similar concentration
of strain 17 was lethal for mice (data not shown).

Internalization of bacterial cells by human PMNLs
In the phagocytosis experiments, strain 17 cells were
rarely internalized, though many of these cells were
bound to the cell surface of PMNLs (Fig. 9A). In contrast,
strain 17-2 cells were readily internalized by PMNLs after
90 min incubation. Many of these bacteria were found in
cytoplasmic vacuoles (Fig. 9B).

Gene expression profiles of strain 17 in biofilm in vitro
We next attempted to compare gene expression patterns of
strain 17 between in biofilm and in planktonic conditions


in vitro. Total RNA was isolated from 12 h cultures of
strain 17 on solid culture media as its biofilm-forming
cells and liquid cultures as planktonic cells, respectively.
When the microarray expression data were compared, a
total of 25 genes were up-regulated by at least four-fold
with statistic significance (p < 0.05) in solid culture con-
dition (Table 4). The expression of several genes which
including those for a levanase (PINA0149), an extracyto-
plasmic function (ECF)-subfamily sigma factor (putative
GcE: PINA0299), a putative lipoprotein (PINA1510), and a
putative polysialic acid transport protein (KpsD,
PINA1911) were protruded. Among hypothetical pro-
teins, PINA1526 (putative CpxP) showed extremely high
levels of transcription.


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Table 3: Genes showing at least two-fold higher expression levels
in biofilm-forming Prevotella intermedia strain 17 than those of
non-forming variant strain 17-2


Gene Fold change Annotation


PIN0258
PIN0281 I
PINA0419
PINA0775
PINA1058
PINA1693
PINA1756
PINA1757
PINA1797
PINA1798
PINA2006


Discussion
It is well known that


Hypothetical protein
Heat shock protein 90, HtpG
Hypothetical protein
Patatin-like phospholipase family protein
DnaK protein
Folylpolyglutamate synthase, FolC
Heat shock protein, DnaJ
Hypothetical protein
Chaperonin, 60 kDa, GroEL
Chaperonin, 10 kDa, GroES
CIpB protein




bacteria assuming biofilm-forming


capacity have enormous advantages in establishing per-
sistent infections even though they appear to be innocu-
ous in their planktonic state [18-20]. Exopolysaccharide
(EPS) is one of the main constituents of the biofilm extra-
cellular matrix [21], and recent investigations have
revealed that each biofilm-forming bacterium produces
distinctive EPS components e.g. alginate and/or Psl found
in Pseudomonas aeruginosa [22], acidic polysaccharide of
Burkholderia cepacia [23], collanic acid, poly-P-1,6-GlcNAc
(PGA) or cellulose found in Escherichia coli [24-27], cellu-
lose of Salmonella [24,28], amorphous EPS containing N-
acetylglucosamine (GlcNAc), D-mannose, 6-deoxy-D-
galactose and D-galactose of Vibrio cholerae [29], polysac-
charide intercellular adhesin (PIA) of Staphylococcus [30],
and glucose and mannose rich components found in
Bacillus subtilis biofilm [31 In this study we found that P.
intermedia strain 17 produced a large amount of EPS, with
mannose constituting more than 80% of the polysaccha-
rides. Among oral bacteria, the production of mannose-
rich polysaccharide by Capnocytophaga ochracea has been
reported [32]. This EPS provides a protection from attack
by the human innate immune system [33]. We have also
reported that a clinical isolate of Prevotella nigrescens can
produce a copious amount of mannose-rich EPS [16].

In this study, biofilm-forming P. intermedia strain 17
showed stronger ability to induce abscesses in mice than
that of strain 17-2, which was a naturally occurring variant
of strain 17 that did not produce surface-associated
fibrous material and therefore not capable of forming a
biofilm. It is evidently shown that the slime/EPS produc-
tion is critical for bacteria to exhibit the resistance to the
neutrophil phagocytosis [33-36], though some EPS are
not essential to bacterial adherence to host cells or for sys-
temic virulence [37,38]. Jesaitis et al. [39] demonstrated
that human neutrophils that settled on P. aeruginosa bio-


films became phagocytically engorged, partially degranu-
lated, and engulfed planktonic bacteria released from the
biofilms. Deighton et al. [40] compared the virulence of
slime-positive Staphylococcus epidermidis with that of
slime-negative strain in a mouse model of subcutaneous
infection and showed that biofilm-positive strains pro-
duced significantly more abscesses that persisted longer
than biofilm-negative strains. TEM observation in our pre-
vious [16] and this study showed that P. nigrescens as well
as P. intermedia with mannose-rich EPS appeared to be
recognized by human leukocytes but not internalized.
Leid et al. [41] have shown that human leukocytes can eas-
ily penetrate Staphylococcus aureus biofilms but fail to
phagocytose the bacteria. Though we have to carefully
investigate the possibility that multiple mutations exist in
strain 17-2 and lead to the observed incapability to induce
abscesses in mice, it is conceivable that biofilm bacteria
being held together by EPS as in this case with strain 17
might present a huge physical challenge for phagocytos-
ing neutrophils. In our previous study [16], we observed
the restoration of the induction of abscess formation in
mice when the purified EPS from the biofilm-forming
strain of P. nigrescens was added to the cultures of a bio-
film-non-forming mutant and injected into mice. As a
consequence of these neutrophils being frustrated by their
inability to phagocytose this bacterial mass, this might
trigger the unregulated release of bactericidal compounds
that could cause tissue injury as shown in the inflamma-
tory pathway associated with lung injury [42,43] or
chronic wounds [44]. The cellular components from neu-
trophils themselves are known to exert a stimulatory effect
on the developing P. aeruginosa biofilm when the host
fails to eradicate the infection [45].

Bacterial biofilm formation is likely to involve a cascade
of gene expression events associating with a crossover of
many sensing systems directed against environmental
changes [46]. When we compare the microarray expres-
sion data obtained from strain 17 as bacterial cells were
producing EPS to those of strain 17-2 as EPS non-produc-
ing variant, stress inducible heat shock proteins were up-
regulated in strain 17 at a gene transcriptional level. We
can not particularize functions of these genes in biofilm
formation of P. intermedia since a genetic transfer system
for having gene-targeted mutants of this organism yet
remains to be developed [47,48]. However, recent studies
evidently showed a tight relation between stress responses
and biofilm formation [46,49-55], though stress response
genes are not prominently up-regulated in some experi-
mental biofilm formation [56]. We found in our earlier
study that exposing biofilm-positive P. intermedia to envi-
ronmental stress such as animal passages of the organism
resulted in the up-regulations of HSPs at a protein level
with increased production of cell surface-associated mesh-
work-like structures. By contrast, animal passages induced


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- Strain 17
0 Strain 17-2


0

0
0


0000
0


U

U


2


E
0
CD)
O
0
0





0


20


F


12h 24h


36h


Figure 5
Growth of P. intermedia strains 1 7 and 1 7-2 in enriched-trypticase soy broth and scanning electron micro-
graphs showing morphological changes associated with growth. Strains 17 and 17-2 entered into the exponential
phase at a different time point. Strain 17-2 shows a faster growth rate (A). Meshwork-like structures around strain 17 cells
were observed at 12 h and became denser with time. Arrowheads indicate cells with meshwork-like structures (B). No such
morphological changes were observed in strain 17-2 (C).


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U
U
0
U p I p


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---- dnaK
Strain 17 A dnaJ
groES
-*- groEL
-*- clpB


if


N

4 4


Figure 6
Validation of the up-regulation of five heat shock pro-
tein genes (PINAI058, PINA1756, PINA1797,
PINA1798, PINA2006) in strain 17 by quantitative
real-time RT-PCR. Total RNA was isolated from 12 h-old
cultures of strains 17 and 17-2, and the expression levels of
these genes were compared by real-time RT-PCR. The aver-
age of increased expression level of PIN 0281 was less than
twofold in the real-time RT-PCR analysis though a three-fold
up-regulation of this gene was observed by the microarray
assay.


neither the production of viscous materials nor the up-
regulation of HSPs in strain 17-2 (unpublished data).

When we compared the gene expression profiles of strain
17 cells plated on BAPs to those of planktonic cells in
enriched-TSB, transcriptional levels of several genes
including those for a levanase (ScrL: PINA0149), putative
GE (PINA0299) and a polysialic acid transport protein
(KpsD: PINA1911) were dramatically up-regulated on
cells from the solid culture media. The highest transcrip-
tional level was observed on a hypothetical protein
(PINA1526) with LTXXQ motif which is found in a
number of bacterial proteins bearing similarity to the pro-
tein CpxP [57]. PINA0299 (putative 7E) is homologous to
the gene for AlgU which affects the conversion to mucoidy
and alginate production in P. aeruginosa [58]. The AlgU
(CE)-dependent promoter of RpoH, well known positive
regulator of heat shock genes, is known to be activated in
mucoid type P. aeruginosa [58]. Although plating of plank-
tonic cells at an exponential phase itself is known to
immediately induce the expression of heat shock regulons
in E. coli [59], we now hypothesize that, like AlgU (cE) in
P. aeruginosa [58], P. intermedia strain 17 cells keep their
stress response via one of ECF sigma factors activated; thus
rendering this organism to maintain EPS production at
high levels in different growth conditions. However, so far


0
()
U)

a-
X

C)
Q)



ir


0


Strain 17-2


6 12 18 24 30 36 h

Culture period

Figure 7
Fluctuation of transcriptional levels of heat shock
protein genes through a culture period in strains 17
and I 7-2. Total RNA was isolated from 6, 12, 18, 24, and 30
h-old cultures of strains 17 and 17-2, and the relative expres-
sion levels of these genes were recorded by the strain using
real-time RT-PCR. The expression levels of these genes were
fluctuating in strain 17 but not in strain 17-2. Data are repre-
sentative of two independent experiments. dnaK: PINA1058;
dnaj: PINA1756; groEL: PINA1797; groES: PINA1798; clpB:
PINA2006.


we studied, gene clusters responsible for mannose-rich
EPS still remain to be elucidated. To address the question
of whether the gene expression phenomena observed in
this study represent gene expression events behind the
EPS production in P. intermedia biofilm, operon/genes for
EPS synthesis regulated by stress-responsive systems of
this organism must be explored in future studies.

Conclusion
The data obtained in this study suggest that the Prevotella
biofilms mainly composed of mannose-rich polysaccha-


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20-



10-


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P. intermedia
strain 17-2


/l j
*.w ^ .:|


Figure 8
Abscess induction in mice. Abscess formation was
induced when 0.5 ml of bacterial cell suspension (3 x 107
CFU/ml) of strain 17 was injected into the inguinal area of a
mouse (left panels). In contrast, the subcutaneous injection of
strain 17-2 (0.5 ml at a concentration of 107 and 108 CFU/ml)
failed to induce an abscess in mice (right panels). Relatively
small abscesses were induced when a higher cell concentra-
tion of strain 17-2 (109 CFU/ml) was injected (right bottom
panel). The data are from one of three independent experi-
ments.


P. intermedia
strain 17


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rides contribute to their resistance to host innate defence
responses resulting in the development of chronic infec-
tions in vivo, and may also suggest that stress responsive
systems of this organism might be behind its biofilm for-
mation. To figure out a biofilm formation-gene expres-
sion relay system in P. intermedia requires the
development of a suitable molecular tool that is capable
of introducing specific targeted mutagenesis on genes
highlighted in this study.

Methods
Bacterial strain and cultures
A viscous material producing clinical isolate of P. interme-
dia, which was isolated from a periodontitis lesion and
designated as strain 17 [12], was used in this study. A total
of 10 frozen culture stocks of isolated strain 17 were used
in this study. Stock cultures of strain 17 in each vial were
grown on trypticase soy blood agar plates (BAP) supple-
mented with 0.5% yeast extract (Difco Laboratories,
Detroit, MI), hemin (5 mg/1), L-cystine (400 mg/1) and
vitamin K1 (10 mg/1) or grown in the enriched-TSB: tryp-
ticase soy broth (TSB; BBL Microbiology Systems, Cockey-
sville, ND) supplemented with 0.5% yeast extract, hemin
(5 mg/1), L-cystine (400 mg/1) and vitamin K1 (10 mg/1).
Bacterial cultures were grown anaerobically in an anaero-
bic chamber (ANX-3, Hirasawa, Tokyo, Japan) at 37 C in
a 5% CO2, 10% H2, 85% N2 atmosphere.

Biofilm phenotype on strain 17 stock cultures
The ability to produce viscous materials in culture media
and form meshwork-like structures on cell surfaces were
used as criteria for distinguishing between "biofilm-form-
ing" and "biofilm-non-forming" as described previously
[16]. We first examined whether strain 17 met the criteria
for being a biofilm-forming bacterium, since more than a
decade has passed when we first described the unique
phenotypic characteristic of strain 17 for its ability to pro-
duce viscous material [12]. Ten culture stocks were plated
on BAP respectively and grown for 48 h anaerobically.
Single colony from each culture stock was transferred to
enriched-TSB and grown for 24 h as the seed culture. One
hundred and fifty gl of this seed culture was transferred to
enriched-TSB (15 ml) and grown for 48 h. The spent cul-
ture medium (550 gl) was put into a rotor, and the viscos-
ity was measured as shearing stress between a rotor and a
rotor shaft at 50 rpm, 20C using a rotary viscometer
(Toki-sangyo, Tokyo, Japan).

To examine cell surface structures, scanning electron
microscopy (SEM) was performed. Bacteria grown on BAP
for 48 h were collected on a piece of filter paper (Glass
fiber GA55, Toyo Roshi, Tochigi, Japan), fixed with 2%
glutaraldehyde in 0.1 M phosphate buffer for 2 h and 1%
OsO4 in 0.1 M phosphate buffer for 1 h at 4 C, and dehy-
drated through an ethanol series and 2-methyl-2-propa-


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4
p
..


sk. inte edia-.
%strain 17 '


A


I
I-,
I.


M.g.,
4"Ohm


;~ i*
.4


0*


C>


.& .

p ,
S*1


Is
4


_ ',*
,.. !


W,. O i

";...- ,


Figure 9
Resistance of viscous material-producing strain 1 7 against the phagocytic activity of human neutrophils. Strain
17 cells were not internalized by neutrophils though many of these cells were bound to the cell surface of neutrophils (A,
arrows). In contrast, viscous material non-producing strain 17-2 cells were internalized and the ingested bacteria appear to be
enclosed within cytoplasmic vacuoles (B, asterisks). Bars = 2.8 inm.




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-i)' C r lo '

qP*-'' ^^ ^


4 m




t .i.. 9 m'
BlB^ '-H'-* *' A


HB.^ -


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Table 4: Genes showing at least four-fold higher expression levels in biofilm-forming Prevotella intermedia strain 17 than those of strain
17 in planktonic condition


Hypothetical protein
Lipoprotein, putative
Levanase, ScrL
Levanase, SacC
Glucose-galactose transporter, putative
Fructokinase
Outer membrane protein
Hypothetical protein
ECF-subfamily sigma factor (CE, putative)
Hypothetical protein
Hypothetical protein
Fibronectin type III domain protein
Hypothetical protein
Ribose-5-phosphate isomerase B
Hemin receptor, putative
Lipoprotein, putative
Hypothetical protein
Hypothetical protein with LTXXQ motif (CpxP, putative)
Hypothetical protein
Cell surface protein
AraC family transcriptional regulator
Polysialic acid transport protein, KpsD
Alkyl hydroperoxide reductase, subunit C, AhpC
Dps protein
Agmatinase, SpeC


nol followed by platinum ion coating (E-1030, Hitachi,
Tokyo, Japan). Specimens were examined with a scanning
electron microscope (S-4800, Hitachi) at an accelerating
voltage of 3 kV.

During the evaluation for the ability of our stock strain 17
cultures to form biofilms, one of the 10 stocks that we
tested was a naturally-occurring variant that lacked the
ability to form biofilms. A stock strain, designated as
strain 17-2, produced neither viscous materials in culture
medium nor cell surface-associated meshwork-like struc-
tures was obtained and considered as a biofilm-negative
variant. These phenotypes were stably maintained in this
variant strain despite repetitive passages in vitro or in ani-
mals (data not shown). The enzymatic activities of strains
17 and 17-2 were examined using the API ZYM system
(bioMerieux, Marcy l'Etoile, France) and there was no sig-
nificant difference regarding the production of enzymes
(data not shown).

Biofilm formation assay
The ability to form biofilm was investigated for strains 17
and 17-2 using crystal violet microtiter plate assay. Briefly,
the seed cultures of both strains were prepared as
described above and diluted to an OD of 0.1 at 620 nm in
the same medium. Next, 150 gil diluted culture was trans-
ferred to each of eight sterile polystyrene microtiter plate


wells (IWAKI, Tokyo, Japan) per strain. Sterile enriched-
TSB was used as a control. The plates were prepared in
duplicate and incubated at 37C for 24 and 48 h, respec-
tively. Biofilm formation was quantified according to
Mohamed et al. [60]. This assay was repeated three times.
A statistical analysis was performed using Student's t-test.

Sugar composition of viscous materials from strain 17
cultures
The exopolysaccharide was prepared from culture super-
natants by the method of Campbell et al. [61]. Briefly, P.
intermedia strain 17 was grown at 37C in enriched-TSB
for 24 h. Supernatants were separated by centrifuging the
liquid culture at 12,000 x g for 30 min, and sodium ace-
tate was added to a final concentration of 5%. The mixture
was stirred for 30 min at 22 C and the exopolysaccharide
was isolated by ethanol precipitation from the reaction
mixture. The ethanol-precipitated material was collected
by centrifugation (18,200 x g for 15 min at 22C),
resolved in 5% sodium acetate, and treated with chloro-
form: 1-butanol (1: 5 by volume). Water-soluble and
chloroform-butanol layer were separated by centrifuga-
tion, an equal amount of ethanol was added to the water-
soluble layer (this procedure was repeated twice), and the
ethanol-precipitated material was freeze-dried and stored
at -80 C until use. Contaminated lipopolysaccharides
(LPS) were removed from preparations according to the



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Gene


Fold change Annotation


PIN0036
PINA0141
PINA0149
PINA0150
PINA0151
PINA0152
PINA0194
PINA0298
PINA0299
PINA0300
PINA0612
PINA0990
PINAI 157
PINA1452
PINA1494
PINAI510
PINA1525
PINA1526
PINA1665
PINA1807
PINA1833
PINA191 I
PINA1931
PINA2066
PINA21 19


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method of Adam et al. [62]. The freeze-dried material was
dissolved in distilled water (0.5 mg/ml), and TritonX- 114
(MP Biomedicals, Eschwege, Germany) stock solution
(lower detergent rich phase) was added to a final concen-
tration of 1% (v/v). After cooling on ice for 30 min, the
solution was stirred at 40C for 30 min and incubated at
370 C until the separation into two layers was complete.
The upper aqueous phase was recovered by centrifugation
for 30 min (1,000 x g, 30 0 C). This Triton X-114 treatment
was performed twice. The upper aqueous phase was
extracted three times with 3 vol CHC13/CH3OH (2: 1 by
volume) to remove detergent. The aqueous phase was
concentrated under reduced pressure and freeze-dried.
The contaminated-LPS level was measured by Limulus
Amebocyte Lysate test according to the manufacturer's
protocol (Seikagaku-kogyo, Tokyo, Japan).

The sugar composition of the purified viscous material
were determined by means of HPLC for neutral and
amino sugars and colorimetry for uronic acid. Briefly,
neutral monosaccharides were released from purified
exopolysaccharide (5 mg) by hydrolysis in a sealed tube
with 2 N trifluoroacetic acid (200 gl) at 1000 C for 6 h. The
hydrolysate was concentrated in vacuo and dissolved in
500 ml of distilled water. The sugars were identified by
HPLC (LC-9A, Shimadzu, Kyoto, Japan) with a TSK-gel
sugar AXG column (15 cm x 4.6 mm) (Tosoh, Tokyo,
Japan) using 0.5 M potassium tetraborate buffer (pH 8.7)
as a carrier at a flow rate of 0.4 ml/min and a column tem-
perature of 70 C. Amino sugars were released from puri-
fied exopolysaccharide (5 mg) by hydrolysis in a sealed
tube with 4 N HC1 (200 gl) at 100 C for 6 h. The hydro-
lysates were analyzed by HPLC (LC-9A, Shimadzu).

Transmission electron microscopy of purified viscous
materials
For negative staining, the ethanol precipitated viscous
material was dissolved in distilled water (1 mg/ml). Fif-
teen microliters of the sample was deposited onto a form-
var-coated and carbon-stabilized copper grid. After 1 min,
excess fluid was removed with filter paper strips, stained
with 2% uranyl acetate for 1 min, and examined in a
transmission electron microscope (TEM) (H7100,
Hitachi, Tokyo, Japan) at 100 kV.

Microarray construction
To create a whole-genome microarray for P. intermedia
strain 17, 30 perfect-matched and 30 miss-matched 24-
mer probes were designed for all putative open reading
frames (ORFs) (2,816 ORFs/array) from a whole genome
sequence of P. intermedia strain 17, which is available
from the Institute for Genomic Research data base (TIGR)
using a Maskless Array Synthesizer (NimbleGen Systems
Inc., Madison, WI, USA).


RNA isolation
To determine an appropriate time point for total RNA iso-
lation from the cultures of strains 17 and 17-2, morpho-
logical changes of cell surface structures associating with
growth were examined by SEM. Single colony of Strains
17 and 17-2 grown on BAP for 24 h were inoculated into
enriched-TSB and grown for 24 h as the seed culture. Five
ml of this seed culture was used to inoculate 500 ml of
enriched-TSB. The growth of the culture was monitored by
measuring the absorbance at the wavelength of 600 nm.
The morphology of cultured cells at a different stage of
growth was examined by SEM as described above. RNA
isolation was performed at a time point (12 h) when the
surface-associated meshwork-like structure had begun to
form. Total RNA samples were extracted from 12 h cul-
tures of strains 17 and 17-2 using RNeasy Midi Kit (QIA-
GEN, Tokyo, Japan) according to the manufacturer's
protocol. Samples were quantified and checked for purity
using an Agilent 2100 bioanalyzer (Agilent, Hachioji,
Japan). Total RNA (12 gg) was primed with random
primer (Invitrogen, Tokyo, Japan), and cDNA was synthe-
sized with reverse transcriptase (Superscript II, Invitro-
gen). The resulting cDNA was fragmented with DNase I
(Promega, Madison, WI, USA) and labeled with biotin
using terminal deoxynucleotidyl transferase (Promega).
Biotin-labeled samples were hybridized onto the strain 17
microarray at 45 C for 16-20 h using NimbleGen's Hybri-
wheel Hybridization chambers (NimbleGen Systems
Inc.).

To compare gene expression profiles of strain 17 in solid
and liquid culture conditions, seed cultures of strain 17
were newly prepared as described above. Five ml of this
seed culture was transferred to enriched-TSB (500 ml) and
200 gl of the seed cultures was transferred to each of 50
BAPs. Both cultures were incubated for 12 h anaerobi-
cally. Total RNA was isolated from the liquid cultures as
described above. Two hundred gl of PBS was added to
BAPs to harvest growing cells using cell scrapers (IWAKI).
Cell suspensions were washed twice with PBS and total
RNA was isolated as described above.

Microarray image acquisitions and data analyses
Hybridized-microarray slides containing technical dupli-
cates were imaged with a high resolution array scanner
(GenePix 4000B Microarray Scanner, Molecular Devices
Corp., Sunnyvale, CA, USA) and the fluorescent signal
intensities from each spot were quantified using NimbleS-
can Software (NimbleGen Systems Inc.). Normalization
was performed among four microarray hybridization data
sets by means of Robust Multi-chip analysis algorithm
[63] and statistical analyses were performed using t-test
and Bonferroni adjustment in the Roche-NimbleGen
Microarray soft wears (Roche Diagnostics, Tokyo, Japan).
When the individual probes met the criteria that the aver-


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age signals from the culture of biofilm-positive strain ver-
sus the average signals from biofilm-negative strain were
different by at least twofold with statistic significance,
probes selected were used to find up-regulated regions.

Pertinent information on raw data containing experimen-
tal designs and hybridization results for specific oligonu-
cleotide sets is available in CIBEX database [17].

Quantitative real-time RT-PCR
To confirm the up-regulation of several genes in strain 17
recorded by the microarray, a real-time RT-PCR strategy
was employed. Twelve hours cultures of strains 17 and 17-
2 were prepared again and total RNA was isolated as
described above. Real-time RT-PCR was performed
according to the one-step RT-PCR protocol of iScriptfM
One-Step RT-PCR Kit with SYBR Green (BIO-RAD Labo-
ratories, Tokyo, Japan). Briefly, 50 ng of total RNA, 200
nM of forward and reverse primers for a target gene, and
25 l of SYBR Green RT-PCR Reaction Mix (BIO-RAD
Laboratories) were added into a PCR tube containing one
gl of iScript Reverse Transcriptase for One-Step RT-PCR.
The PCR preparation was brought to a final volume of 50
gl with nuclease-free water (BIO-RAD Laboratories). As an
internal control, RT-PCR for 16S rRNA was performed at
50C for 10 min, 95C for 5 min, followed by 35 cycles
at 95 oC for 10 sec and 640C for 30 sec followed by melt
curve analysis. Primers for 16S rRNA and several genes
showing up-regulation in microarray assays were as fol-
lows: 16S rRNA: forward 5'-AGAGTTFGATCCTGGCT-
CAG-3', reverse 5'-AAAGGAGGTGATCCAGCC-3'; PIN
0281: forward 5'-TGAACGTAAGCCGCAGCTAC-3',
reverse 5'-TTGTTCTTGGCGCAAAGCAG-3'; PINA1058:
forward 5'-TGTGAACCCCGACGAAGTGG-3', reverse 5'-
GTGCCTGCTGACCAGCATCT-3'; PINA1756: forward 5'-
AATACAGCCTTCGAGGGTIT-3', reverse 5'-TTCGGTCAA-
GACAGTAGGGA-3'; PINA1797: forward 5'-TGAAGATIT-
GCGCTGTCAAG-3', reverse 5'-
TAGCAGGAGTITCITCAGGT-3'; PINA1798: forward 5'-
AGCGGAGCAGAAAGTAGGTG-3', reverse 5'-CAACAG-
CAAGAACGTCGCTT-3'; PINA2006: forward 5'-CIT-
GGAACAACGGGTACAGG-3', reverse 5-
AAATCTCGCITTGCGTCAGT-3'. Annealing temperatures
were optimized for each primer pair by the use of melting
curve analysis in which the melting curve starts at 55 C
and ends at 900C with temperature increment of 0.20 C
and a hold time of 2 sec. The optimized annealing temper-
ature for each target gene was 64.50C for PIN 0281,
62.00C for PINA1058, 64.5 C for PINA1756, 65.0Cfor
PINA1797, 58.70C for PINA1798 and 57.60C for
PINA2006, respectively. The threshold cycle (Gr) values
were obtained for the reactions reflecting the quantity of
the template in the sample. ACr for each gene was calcu-
lated by subtracting the calibrator gene 16S rRNA QCvalue
from each of the target values represented the relative


quantity of the target mRNA normalized to the level of the
internal standard 16S rRNA mRNA level. The target
mRNA levels in strains 17 and 17-2 were defined and
compared.

To observe how the expression levels of these genes fluc-
tuate through the culture period, single colony of strains
17 and 17-2 grown on BAP for 24 h were inoculated into
enriched-TSB and grown for 24 h as the seed culture. One
hundred and fifty gl of this seed culture was used to inoc-
ulate 15 ml of enriched-TSB. Total RNA samples were
extracted from 6, 12, 18, 24 and 30 h cultures of strains 17
and 17-2 using RNeasy Midi Kit (QIAGEN) and applied to
the real-time RT-PCR as described above. Changes of the
target mRNA levels through the culture period were
recorded by the strain.

Animal studies
The virulence of biofilm-forming strain 17 was compared
with that of biofilm-non-forming variant strain 17-2
regarding abscess formation in mice. Bacterial strains were
cultured in enriched-TSB for 24 h for strain 17-2 and 36 h
for strain 17, respectively (early stationary phase; see Fig.
5). Five hundred gl of bacterial suspensions (106 to 1010
CFU/ml) was injected subcutaneously into the inguen of
each BALB/c mouse (male, 4 weeks; 3 mice per strain).
Changes of abscess lesions were recorded photographi-
cally using a camera (Nikon FillJI, Nikon, Japan) set at a
fixed magnification for five consecutive days.

Phagocytosis assay
To compare anti-phagocytic activity of strain 17 with that
of strain 17-2, bacterial cells were co-cultured with poly-
morphonuclear leukocytes (PMNL) obtained from
healthy human volunteers (n = 3; age 20-23 years) in
accordance with institutional approved procedures.
PMNLs were purified by a PolymorphprepTM (Axis-
Shield PoC AS, Oslo, Norway) gradient and cultured in
RPMI 1640 medium (Gibco BRL, Grand Island, NY) sup-
plemented with 10% heat-inactivated fetal calf serum
(FCS) (MultiSer, Trace Scientific Ltd, Melbourne, Aus-
tralia) and 2 mM L-glutamine at 37C in 5% CO2. Bacte-
rial cell suspensions (1.5 x 106 CFU/ml) were prepared
from strains 17 and 17-2 cultures as described in the ani-
mal studies. Three hundred gl of PMNLs (106 cells/ml)
was dispensed into the wells of 24-well tissue culture
plates (Becton Dickinson, Franklin Lakes, NJ). To these
wells, 100 gl of bacterial suspension of different tested
strains was added. After incubation for 60-90 min at
37 oC, PMNLs co-cultured with bacterial cells were centri-
fuged at 8,000 x g at 40C for 5 min and processed for
transmission electron microscopy to determine the inter-
nalization of tested strains by PMNLs. Cell pellets were
fixed with 2% glutaraldehyde in 0.1 M phosphate buffer
for 2 h at 4C, post-fixed with 1% OsO4 in 0.1 M phos-


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phate buffer for 1 h at 4oC, and dehydrated through an
ethanol series. Samples were embedded into Epon resin
and ultrathin sections were prepared by a ultramicrotome
(Ultracut, Leica, Tokyo, Japan). Ultrathin sections were
placed on a copper grid, stained with uranyl acetate and
lead citrate, and observed in a TEM (H7100, Hitachi).


Authors' contributions
TY, TF and CM carried out the phenotype characterization
and microarray analysis, and drafted the manuscript. KY
and CS performed RT-PCR. NM and HN screened a cul-
ture collection of strain 17 for the ability to produce vis-
cous material. TN participated in the analysis of
microarray data. CBW, KPL, and HF participated in the
design of this study and drafted the manuscript.


Acknowledgements
We would like to acknowledge Mr. Hideaki Hori for his excellent assistance
with the electron microscopy. Part of this study was performed at the Insti-
tute of Dental Research, Osaka Dental University. This study was sup-
ported in part by Osaka Dental University Joint Research Fund (B08-01).

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