Group Title: BMC Microbiology
Title: Distinct roles of long/short fimbriae and gingipains in homotypic biofilm development by Porphyromonas gingivalis
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Title: Distinct roles of long/short fimbriae and gingipains in homotypic biofilm development by Porphyromonas gingivalis
Physical Description: Book
Language: English
Creator: Kuboniwa, Masae
Amano, Atsuo
Hashino, Ei
Yamamoto, Yumiko
Inaba, Hiroaki
Hamada, Nobushiro
Nakayama, Koji
Tribble, Gena
Lamont, Richard
Shizukuishi, Satoshi
Publisher: BMC Microbiology
Publication Date: 2009
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Abstract: BACKGROUND:Porphyromonas gingivalis, a periodontal pathogen, expresses a number of virulence factors, including long (FimA) and short (Mfa) fimbriae as well as gingipains comprised of arginine-specific (Rgp) and lysine-specific (Kgp) cysteine proteinases. The aim of this study was to examine the roles of these components in homotypic biofilm development by P. gingivalis, as well as in accumulation of exopolysaccharide in biofilms.RESULTS:Biofilms were formed on saliva-coated glass surfaces in PBS or diluted trypticase soy broth (dTSB). Microscopic observation showed that the wild type strain formed biofilms with a dense basal monolayer and dispersed microcolonies in both PBS and dTSB. A FimA deficient mutant formed patchy and small microcolonies in PBS, but the organisms proliferated and formed a cohesive biofilm with dense exopolysaccharides in dTSB. A Mfa mutant developed tall and large microcolonies in PBS as well as dTSB. A Kgp mutant formed markedly thick biofilms filled with large clumped colonies under both conditions. A RgpA/B double mutant developed channel-like biofilms with fibrillar and tall microcolonies in PBS. When this mutant was studied in dTSB, there was an increase in the number of peaks and the morphology changed to taller and loosely packed biofilms. In addition, deletion of FimA reduced the autoaggregation efficiency, whereas autoaggregation was significantly increased in the Kgp and Mfa mutants, with a clear association with alteration of biofilm structures under the non-proliferation condition. In contrast, this association was not observed in the Rgp-null mutants.CONCLUSION:These results suggested that the FimA fimbriae promote initial biofilm formation but exert a restraining regulation on biofilm maturation, whereas Mfa and Kgp have suppressive and regulatory roles during biofilm development. Rgp controlled microcolony morphology and biovolume. Collectively, these molecules seem to act coordinately to regulate the development of mature P. gingivalis biofilms.
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Research article


Distinct roles of long/short fimbriae and gingipains in homotypic
biofilm development by Porphyromonas gingivalis
Masae Kuboniwa*1, Atsuo Amano2, Ei Hashino1, Yumiko Yamamoto1,
Hiroaki Inaba2, Nobushiro Hamada3, Koji Nakayama4, Gena D Tribble5,
Richard J Lamont6 and Satoshi Shizukuishi1


Address: 'Department of Preventive Dentistry, Osaka University Graduate School of Dentistry, Suita-Osaka, Japan, 2Department of Oral Frontier
Biology, Osaka University Graduate School of Dentistry, Suita-Osaka, Japan, 3Department of Oral Microbiology, Kanagawa Dental College,
Yokosuka-Kanagawa, Japan, 4Department of Developmental and Reconstructive Medicine, Nagasaki University Graduate School of Biomedical
Sciences, Nagasaki, Japan, 5Department of Periodontics, The University of Texas Health Science Center at Houston, Houston, TX, USA and
6Department of Oral Biology, University of Florida College of Dentistry, Gainesville, FL, USA
Email: Masae Kuboniwa* kuboniwa@dent.osaka-u.ac.jp; Atsuo Amano amanoa@dent.osaka-u.ac.jp; Ei Hashino hashino@dent.osaka-
u.ac.jp; Yumiko Yamamoto y-ymmt@dent.osaka-u.ac.jp; Hiroaki Inaba hinaba@dent.osaka-u.ac.jp;
Nobushiro Hamada hamadano@kdcnet.ac.jp; Koji Nakayama knak@nagasaki-u.ac.jp; Gena D Tribble gena.d.tribble@uth.tmc.edu;
Richard J Lamont rlamont@dental.ufl.edu; Satoshi Shizukuishi shizuku@dent.osaka-u.ac.jp
* Corresponding author



Published: 26 May 2009 Received: 26 December 2008
BMC Microbiology 2009, 9:105 doi:10.1 186/1471-2180-9-105 Accepted: 26 May 2009
This article is available from: http://www.biomedcentral.com/1471-2180/9/105
2009 Kuboniwa et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.ore/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.



Abstract
Background: Porphyromonas gingivalis, a periodontal pathogen, expresses a number of virulence factors,
including long (FimA) and short (Mfa) fimbriae as well as gingipains comprised of arginine-specific (Rgp) and
lysine-specific (Kgp) cysteine proteinases. The aim of this study was to examine the roles of these
components in homotypic biofilm development by P. gingivalis, as well as in accumulation of
exopolysaccharide in biofilms.
Results: Biofilms were formed on saliva-coated glass surfaces in PBS or diluted trypticase soy broth
(dTSB). Microscopic observation showed that the wild type strain formed biofilms with a dense basal
monolayer and dispersed microcolonies in both PBS and dTSB. A FimA deficient mutant formed patchy
and small microcolonies in PBS, but the organisms proliferated and formed a cohesive biofilm with dense
exopolysaccharides in dTSB. A Mfa mutant developed tall and large microcolonies in PBS as well as dTSB.
A Kgp mutant formed markedly thick biofilms filled with large clumped colonies under both conditions. A
RgpA/B double mutant developed channel-like biofilms with fibrillar and tall microcolonies in PBS. When
this mutant was studied in dTSB, there was an increase in the number of peaks and the morphology
changed to taller and loosely packed biofilms. In addition, deletion of FimA reduced the autoaggregation
efficiency, whereas autoaggregation was significantly increased in the Kgp and Mfa mutants, with a clear
association with alteration of biofilm structures under the non-proliferation condition. In contrast, this
association was not observed in the Rgp-null mutants.
Conclusion: These results suggested that the FimA fimbriae promote initial biofilm formation but exert
a restraining regulation on biofilm maturation, whereas Mfa and Kgp have suppressive and regulatory roles
during biofilm development. Rgp controlled microcolony morphology and biovolume. Collectively, these
molecules seem to act coordinately to regulate the development of mature P. gingivalis biofilms.



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Background
Porphyromonas gingivalis has been shown to be a major eti-
ologic agent of destructive adult periodontitis, with a sig-
nificant lifestyle component harbored within the complex
multi-species biofilm (dental plaque) that develops along
the gingival margins [1]. The bacterium expresses a
number of potential virulence factors, such as long
(major) and short (minor) fimbriae, lipopolysaccharides
(LPS), and proteases [2]. Among these factors, a unique
class of cysteine proteinases, termed gingipains, com-
posed of arginine-specific [Arg-gingipain A and B, (RgpA
and RgpB, respectively)] and lysine-specific (Kgp) pro-
teases, are implicated in a wide range of both pathological
and physiological processes [3]. Proteases can be post-
translationally processed for retention on the cell surface
or secretion into the extracellular milieu. Rgp enzymes are
glycosylated, with their carbohydrate domain containing
phosphorylated branched mannans that can contribute to
the anchoring of Rgp on bacterial outer membrane [4]. In
addition, this phosphorylated branched mannan consti-
tutes an exopolysaccharide that is distinguishable from
both LPS and the serotypeable capsule polysaccharides of
P. gingivalis [4].

The cell-associated gingipains comprise the majority
(~80%) of Rgp and Kgp activities, and are reported to be
definitive virulence factors that degrade various host pro-
teins, leading to impaired cellular integrity and function
[5]. In addition, gingipains can mediate bacterial interac-
tions with host components [6]. Recent findings indicate
that gingipains are also involved in biofilm development.
Polyphenolic inhibitors of gingipains can prevent not
only homotypic (monospecies) biofilm formation by P.
gingivalis [7], but also synergistic biofilm formation with
Fusobacterium nucleatum [8]. In addition, an RgpB-defi-
cient mutant of P. gingivalis lost the ability to form syner-
gistic biofilms with Treponema denticola [9]. A low
molecular weight tyrosine phosphatase, Ltpl, was found
to be involved in biofilm formation via suppression of
exopolysaccharide production and luxS expression, as
well as dephosphorylation of gingipains [10]. Thus, gingi-
pains and gingipain regulation may be related to exopol-
ysaccharide accumulation. However, the exact role of
gingipains in biofilm development remains to be eluci-
dated.

Two distinct fimbria types, long and short fimbriae, are
present on the surface of P. gingivalis cells [111. Long fim-
briae impact the host immune response by inducing
human peripheral macrophages and neutrophils to over-
produce several proinflammatory cytokines such as inter-
leukin-1 (IL-1), IL-6, and tumor necrosis factor alpha,
through coordinated interactions with pattern-recogni-
tion receptors [12]. Long fimbriae were also reported to
induce cross-talk between CXC chemokine receptor 4 and


Toll-like receptor 2 in human monocytes and thus under-
mine host defense [13]. Furthermore, long fimbriae are
prominent adhesins that mediate colonization in perio-
dontal tissues and invasion of host cells as well as dysreg-
ulation of host cell cycle, which assists P. gingivalis in its
persistence in periodontal tissues [14,15]. While, the role
of short fimbriae in virulence is less well understood, they
are necessary for the development of synergistic biofilms
between P. gingivalis and Streptococcus gordonii via a spe-
cific interaction with the streptococcal SspB protein [16].
Recently, these two distinct types of fimbriae were
reported to function cooperatively in the development of
homotypic biofilms of P. gingivalis [17]. It was proposed
that the long fimbriae were responsible for bacterial
attachment to the substrate as well as initiation of coloni-
zation, whereas short fimbriae were involved in the for-
mation of microcolonies and biofilm maturation. In that
study, it was also shown that short fimbriae promoted
bacterial autoaggregation, which was suppressed by the
long fimbriae. In contrast, another study showed opposite
results, as deletion of short fimbriae enhanced autoaggre-
gation and negligible autoaggregation occurred in the
long fimbria mutants tested [18]. Thus, the contextual
roles of these fimbria types in biofilm development are
unclear, and further study is necessary.

In the present study, we examined the roles of long and
short fimbriae as well as Arg-and Lys-gingipains in homo-
typic biofilm formation by P. gingivalis using a series of
deletion mutants of strain ATCC33277.

Results
Microstructure of biofilms under nonproliferation
condition
First, we evaluated the roles of long/short fimbriae and
gingipains in initial attachment and organization of bio-
films which is a crucial event in the early phase of biofilm
formation [19]. When cultured in TSB as free-living cells,
wild type and all mutant strains showed the similar
growth rates, as reported in previous study [20]. In con-
trast, when incubated in PBS for 24 h, wild type and
mutants lacking long and/or short fimbriae formed dis-
tinct biofilms (Figure 1 and Table 1). Wild type strain
33277 formed biofilms with a dense basal monolayer and
dispersed microcolonies. Compared with the wild type,
the long fimbria mutant KDP150 formed patchy and
sparser biofilms with a significantly greater distance
between fewer peaks, although mean peak height was
almost the same as that of the wild type strain. In contrast,
the short fimbria mutant MPG67 developed cluster and
channel-like biofilms consisting of significantly taller
microcolonies compared to the wild type. Similar to
MPG67, the mutant (MPG4167) lacking both types of
fimbriae also formed thick biofilms with significantly
taller microcolonies than the wild type. Viability of the


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ATCC33277
(WT)


KDP150
(FimA)


MPG67
(Mfal)


MPG4167
/fimA AMf1 -


(189.4 pm)
Y


x 1ioy.' pm) x x x

(22.1 pm) (16.8 pm) (29.8 pm) (33.2 pm)


KDP129
(Kan)


KDP133
(RoDA RaDI


KDP136


(44.7 pm) (45.9 pm)


(35.4 pm)


Figure I
Homotypic biofilm formation by P. gingivalis wild-type strain and mutants in PBS. P. gingivalis strains were stained
with CFSE (green) and incubated in PBS for 24 hours. After washing, the biofilms that developed on the coverglass were
observed with a CLSM equipped with a 40x objective. Optical sections were obtained along the z axis at 0.7-pm intervals, and
images of the x-y and x-z planes were reconstructed with an imaging software as described in the text. Upper panels indicate z
stacks of the x-y sections. Lower panels are x-z sections. P. gingivalis strains used in this assay are listed in Table 4. The experi-
ment was repeated independently three times with each strain in triplicate. Representative images are shown.


cells in biofilms of each strain was tested by colony count
and confirmed at 24 h (data not shown). These results
suggest that the long fimbriae are involved in initial
attachment and organization of biofilms by P. gingivalis,
whereas the short fimbriae have a suppressive regulatory
role for these steps.

The involvement of gingipains in biofilm formation was
evaluated using a set of P. gingivalis mutants lacking Kgp
(KDP129), RgpA/B (KDP133), or both Kgp and RgpA/B
(KDP136). These mutants lacked the proteolytic domains
as well as the adhesion domains of gingipains [5]. In addi-
tion, both Rgp mutants (KDP133 and KDP136) lacked
bacterial cell-surface structural components such as long
and short fimbriae and hemagglutinins which are proc-
essed by Rgp [21-23]. The Kgp mutant KDP129 formed
markedly thick biofilms containing large accumulations
of which the mean height was significantly taller than the
wild type (Figure 1 and Table 1). In addition, the effi-
ciency of autoaggregation in KDP129 was significantly
increased (Table 2). These results suggest that Kgp plays a


negative role in biofilm development via suppressing
autoaggregation and/or regulating dispersion, de-concen-
tration, and/or detachment of microcolonies. The RgpA/B
mutant KDP 133 formed channel-like biofilms with fibril-
lar microcolonies (Figure 1), which featured significantly
fewer peaks and longer distances between peaks, but
increased height, as compared to those of the wild type
and Kgp mutant (Table 1). Although the features of
KDP133 were likely attributable to the loss of multiple
factors on the bacterial surface, Rgp itself might be a
bifunctional mediator promoting peak formation and
shearing the fibrillar microcolonies of biofilms. Interest-
ingly, the biofilms formed by the gingipain null mutant
(KDP136) showed different features from both the Kgp
(KDP129) and Rgp (KDP133) mutants. Although the
three mutants, KDP136, KDP133 and MPG4167, resem-
ble each other in terms of lack of expression of both types
of fimbriae, their microstructures were divergent (Figure
1). These findings suggested that biofilm formation was
affected not only by the post-translational regulation of
the expression of cell surface components by Rgp, but also


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Table I: Features of biofilms formed by P. gingivalis wild-type strain and mutants in PBS

Peak parameters)


Number of peaks


Mean distance between peaks (pm)


Mean peak height (ptm)


ATCC33277
(wild type)
KDP 50
(AfimA)
MPG67
(Amfal)
MPG4167
(AfimAAmfa I)
KDP129
(Akgp)
KDP133
(ArgpAArgp8)
KDP136
(ArgpAArgpBAkgp)


28.5 3.3

14.7 2.4**

29.3 2.0

30.5 1.9

25.5 2.1

13.0 2.6**

30.5 2.4


3.0 0.2

5.4 1.0.*

3.6 0.2

3.1 0.2

3.6 0.3

8.4 1.3.*

3.2 0.2


a) Number of peaks was evaluated in an area sized 90 (x axis) x 2 (y axis) pm. The mean SE of 10 areas was shown.
**p < 0.01 in comparison with the wild type using a Scheffe test.


by uncharacterized steps that were not altered by Rgp.
Loss of all gingipain activities might result in downstream
events which did not happen in KDP129 and KDP133.

Quantitative analysis of biofilms in PBS
The biovolume of the biofilms was also altered by dele-
tion of various bacterial factors (Figure 2). The deletion of
long fimbriae significantly reduced the biovolume,
whereas the mutant without short fimbriae developed
extensive biofilms. The deletion of Kgp also increased the
biovolume, whereas no significant change was observed
in the Rgp mutants. These results support the above sug-

Table 2: Autoaggregation of P. gingivalis wild-type strain and
mutants


Strain


Autoaggregation index)
(-dA/min)


ATCC33277
(wild type)
KDP 150
(AfimA)
MPG67
(Amfa l)
MPG4167
(AfimAAmfa I)
KDP 129
(Akgp)
KDP133
(ArgpAArgp8)
KDP136
(ArgpAArgp8Akgp)


17.73 1.67

0.54 3.94**

36.12 2.40**

33.87 2.77**

35.62 2.52**

15.04 2.68

0.29 3.22**


a) dA/min was automatically calculated by subtraction of At, the
absorbance at time t min, from At+, at time (t + I) min during
incubation. The maximum value of- dA/min in a curve was used as
the autoaggregation index. The data represent the mean SE of three
separate experiments with each strain in duplicate.
**p < 0.01 in comparison with the wild type using a Scheffe test.


gested roles; i.e., long fimbriae are a facilitator, short fim-
briae and Kpg are suppressors, whereas Rgp has dual
functions, promoting peak formation and shearing the
fibrillar microcolonies, in the initial phase of biofilm for-
mation by P. gingivalis.

Microstructure under proliferation condition
Next, the roles of the fimbriae and gingipains were exam-
ined in the early maturation phase of biofilms, which is
associated with an increase in biovolume mainly due to
cell division and exopolysaccharide accumulation. Bio-
film development was induced by culture in nutrient
medium. Figure 3 shows various features of biofilms of
the mutants incubated in dTSB for 24 hours. The wild type
strain formed biofilms with a dense basal monolayer with
dispersed microcolonies, similar to the PBS condition, but
with more and taller peaks (Table 3). The long fimbria
mutant KDP150 formed biofilms with a thicker monol-
ayer and with a greater number of the fine, taller peaks
compared to wild type, (Figure 3 and Table 3). Those fea-
tures suggested that long fimbriae have a role in suppres-
sion of the development of an thickened basal layer, but
trigger protruding peak formation in early maturation
phase. The short fimbria mutant MPG67 formed signifi-
cantly clustered biofilms consisted of tall and wide micro-
colonies, suggesting that short fimbriae negatively control
the morphology of microcolonies, as mentioned above.
The mutant lacking both types of fimbriae (MPG4167)
also formed markedly thick and dense biofilms contain-
ing various size of microcolonies, suggesting that both
types of fimbriae negatively regulate biofilm formation in
early maturation phase. The Kgp mutant KDP129 formed
large microcolonies which were well dispersed, whereas
the Rgp mutant KDP133 made the most thick biofilms
with the tallest acicular microcolonies (Figure 3 and Table


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Strain


2.8 0.4

2.7 0.8


16.6 0.8**

12.7 0.5**

12.7 1.3.*

23.2 2.8**

12.7 0.7**


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33277 (WT)

KDP150 (FimA)

MPG67 (Mfal)

MPG4167 (FimA Mfal)

KDP129 (Kgp)

KDP133 (RgpA RgpB)

KDP136 (RgpA RgpB Kgp)


MEU-


-~


1.0E+07 2.0E+07 3.0E+07 4.0E+07
Biovolume (pm3)

Figure 2
Quantification of homotypic biofilms formed by P. gingivalis wild-type strain and mutants in PBS. Biofilms were
formed as described in Figure I, and 10 fields per a sample were randomly recorded and quantified with a CLSM. Z stacks of
the x-y sections were converted to composite images to quantify each biovolume as described in the text. Standard error bars
are shown. Statistical analysis was performed using a Scheffe test. *p < 0.05 and **p < 0.01 in comparison to the wild-type
strain. P. gingivalis strains used in this assay are listed in Table 4.


3). These findings suggested that Kgp suppresses micro-
colony expansion, whereas Rgp mediates transverse
enlargement and restrains the longitudinal extension. As
with the result in PBS, biofilms with the gingipain null
mutant KDP136 showed different features from both
KDP129 and KDP133.

Quantitative analysis of biofilms in dTSB
In the early maturation phase, the biovolumes of the bio-
films were significantly increased in all of tested mutants
as compared to the wild type (Figure 4). Deletion of long
fimbriae resulted in the opposite tendency from the initial


attachment phase, suggesting that this molecule has dis-
tinct roles under the different phases.

Exopolysaccharide production under proliferation
conditions
As extracellular polysaccharide is important for the devel-
opment of biofilm communities, we examined the influ-
ences of fimbriae and gingipains on the accumulation of
exopolysaccharide in P. gingivalis biofilms. To visualize
and quantify exopolysaccharide accumulation in biofilms
under the proliferation condition, 4',6-diamino-2-phe-
nylindole (DAPI)-labeled P. gingivalis cells and fluores-


Table 3: Features of biofilms formed by P. gingivalis wild-type strain and mutants in dTSB

Peak parameters)


Number of peaks


Mean distance between peaks (ptm)


Mean peak height (ptm)


ATCC33277
(wild type)
KDP 150
(AfimA)
MPG67
(Amfal)
MPG4167
(AfimAAmfa I)
KDP129
(Akgp)
KDP133
(ArgpAArgp8)
KDP136
(ArgpAArgpbAkgp)


45.5 3.5

52.5 3.5*

35.8 3.6**

32.3 3.8**

39.8 3.2

41.0 5.7

43.0 1.4


2.0 0.9

1.7 0.7*

2.7 1.6.*

3.0 1.6.*

2.2 1.2

2.2 1.0

2.1 0.8


6.9 1.4


23.7 5.6**

20.9 4.4**

20.5 4.3**

19.6 5.4**

45.9 4.5**

22.2 2.4**


a)Number of peaks was evaluated in an area sized 90 (x axis) x 2 (y axis) pm. The mean SE of 10 areas was shown.
*p < 0.05 and **p < 0.01 in comparison with the wild type using a Scheffe test.

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Strain


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ATCC33277
(WT)


KDP150
(FimA)


MPG67
(Mfal)


MPG4167


(189.4 pm)




x(189.4 pm) x x x

(25.6 m) (35.2 m) (36.6 m) (58.1 z m)
(25.6 pm) (35.2 pm) (36.6 pm) (58.1 pm)


KDP129
(Kgp)


KDP133
(RgpA RgpB)


(48.3 pm) (81.3 pm)


KDP136
(RgpA RgpB Kgp)
4


7 x



(37.6 pm)


Figure 3
Homotypic biofilm formation by P. gingivalis wild-type strain and mutants in dTSB. P. gingivalis strains were stained
with CFSE (green) and incubated in dTSB for 24 hours. After washing, the biofilms that developed on the coverglasses were
observed with a CLSM equipped with a 40x objective. Optical sections were obtained along the z axis at 0.7-prm intervals, and
images of the x-y and x-z planes were reconstructed with imaging software, as described in the text. Upper panels indicate z
stacks of the x-y sections. Lower panels show x-z sections. The experiment was repeated independently three times with each
strain in triplicate. Representative images are shown.


cein isothiocyanate (FITC)-labeled exopolysaccharide
were examined by confocal microscopy with digitally
reconstructed image analysis. In all of the tested strains,
DAPI-labeled cells exhibited the same microstructures of
biofilms composed of 5-(and-6)-carboxyfluorescein suc-
cinimidyl ester (CFSE)-labeled cells, as shown in Figure 3,
thus validating the use of these live-staining methods
(data not shown). Exopolysaccharide visualization ena-
bled us to assess the accumulation pattern (Figure 5A) and
exopolysaccharide biovolume per base area (Figure 5B).
Furthermore, the exopolysaccharide production was nor-
malized to the levels of DAPI-labeled P. gingivalis cells in
the biofilms and expressed as the exopolysaccharide/cell
ratio (Figure 5C). Interestingly, a unique pattern of
exopolysaccharide accumulation was observed in the Rgp
mutant KDP 133 in vertical sections (x-z plane) ofbiofilms
(Figure 5A). In contrast to the other strains, exopolysac-
charide accumulated in the middle layer, and the biofilm
surface was not covered with exopolysaccharide. It was
also notable that the long fimbria mutant KDP150 devel-


oped a biofilm enriched with exopolysaccharide (Figure
5A), reflecting a significantly higher exopolysaccharide/
cell ratio (Figure 5C). The gingipain null mutant KDP136
produced the most abundant exopolysaccharide per unit
base area (Figure 5B). The minor fimbria mutant MPG67,
long/short fimbriae mutant MPG4167 and Rgp mutant
KDP 133 also accumulated significantly larger amounts of
exopolysaccharide than wild type; however, exopolysac-
charide/cell ratio in KDP133 and MPG4167 was signifi-
cantly lower than wild type because biofilms of these
strains consisted of larger numbers of cells (Figure 5C).

Autoaggregation
Bacterial autoaggregation has been reported to play an
important role in initial biofilm formation [24], thus the
autoaggregation efficiencies of the mutants were assessed
(Table 2). Deletion of long fimbriae significantly reduced
the autoaggregation efficiency, which agreed with the pre-
vious report that long fimbriae were required for autoag-
gregation [25]. The efficiency of autoaggregation was


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33277 (WT)

KDP150 (FimA)

MPG67 (Mfal)

MPG4167 (FimA Mfal)

KDP129 (Kgp)

KDP133 (RgpA RgpB)

KDP136 (RgpA RgpB Kgp)


-I


1.0E+07 2.0E+07 3.0E+07 4.0E+07
Biovolume (pm3)

Figure 4
Quantification of homotypic biofilms formed by P. gingivalis wild-type strain and mutants in dTSB. Biofilms were
formed as described in the legend to Figure 3, and 10 fields per a sample were randomly recorded and quantified, similar to the
method described in the legend to Figure 2. Statistical analysis was performed with a Scheffe test. *p < 0.05 and **p < 0.01 in
comparison to the wild-type strain.


significantly increased in the Kgp mutant KDP129, short
fimbria mutant MPG67 and long/short fimbriae deficient
mutant MPG4167, suggesting that Kgp and short fimbriae
act to suppress autoaggregation. Contrary to our predic-
tion, the gingipain null mutant KDP136 and Rgp mutant
KDP133 showed different tendencies of autoaggregation
from MPG4167, although all of these strains were consid-
ered to be long/short fimbriae deficient mutants. Thus,
not only fimbrial expression but also other factors, modi-
fled by gingipains, seem to be involved in autoaggrega-
tion. In addition, it was found that autoaggregation and
biofilm parameters such as biovolume, number of peaks
and peak height were not significantly correlated in every
strain (Figure 2, Figure 4, Table 1 and Table 3). This result
suggests that autoaggregation is not the sole determinant
of alteration in structure of P. gingivalis biofilms.

Tenacity of biofilms
To analyze the influence of the molecules under investiga-
tion on vulnerability of biofilms, the physical strength of
the biofilms against brief ultrasonication was compared
(Figure 6). Consistent with the results of image analysis
described in Figure 4 and Figure 5A, the long/short fim-
briae mutant MPG4167 and Rgp mutant KDP133 formed
expansive biofilms with large numbers of cells in dTSB,
however, their strength was found to be very fragile com-
pared to the other strains, suggesting that these biofilms
consisted of loosely connected microcolonies. In contrast,
the biofilms of the long fimbria mutant KDP150 were
resistant to sonic disruption, suggesting that long fimbriae
are initial mediator of biofilm formation but are not


required to maintain resistance against environmental
shear force.

Collectively, these results suggest that long fimbriae are
required for initial formation of biofilms by P. gingivalis,
but suppress the development of an exopolysaccharide-
enriched basal layer that is related to the adhesive prop-
erty of biofilms. In contrast, short fimbriae and Kgp may
have suppressive and regulatory roles for biofilm forma-
tion, with control over morphology of microcolonies,
whereas Rgp mediates microcolony formation and
restrains the biovolume. In addition, other factors beside
fimbriae and gingipains are likely involved in homotypic
biofilm formation by P. gingivalis.

Discussion
Dental plaque, a precursor for periodontal disease, is also a
well studied model of bacterial biofilms in general [26,27].
Developing biofilm communities in the oral cavity are fun-
damental for the persistence of organisms such as P. gingiva-
lis and continual exposure of the host to P. gingivalis can
result in a dysfunctional immune response [28]. Biofilm
maturation proceeds through a series of developmental steps
involving the attachment of cells to, and growth on, a sur-
face, followed by detachment and dissemination to a new
site to start the cycle again [29,30]. It is likely that much of
biofilm-specific physiology is devoted to dynamic changes
that both stimulate an increase in biovolume and limit or
stabilize accumulation according to environmental con-
straints. Therefore, multiple bacterial factors are thought to
be required to regulate appropriate biofilm structure.


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33277 (WT)
KDP150 (FimA)
MPG67 (Mfal)
MPG4167 (FimA Mfal)
KDP129 (Kgp)


KDP133 (RgpA RgpB)


KDP136 (RgpA RgpB Kgp)


1) 33277 (WT)
KDP150 (FimA)
MPG67 (Mfal)
MPG4167 (FimA Mfal)
KDP129 (Kgp)
KDP133 (RgpA RgpB)
KDP136 (RgpA RgpB Kgp)


.') 33277 (WT)
KDP150 (FimA)
MPG67 (Mfal)
MPG4167 (FimA Mfal)
KDP129 (Kgp)
KDP133 (RgpA RgpB)
KDP136 (RgpA RgpB Kgp)


mentioned dynamic changes that stimulate, limit or stabilize
the biofilm formation. Long fimbriae were shown to be ini-
tial positive mediators of biofilm formation, however, these
appendages also functioned to decrease the adhesive prop-
erty of biofilms via repressing exopolysaccharide accumula-
tion in basal layer. In addition, short fimbriae as well as Kgp
were found to be negative regulators of microcolony forma-
tion and of biovolume. Rgp seems to play a bifunctional role
in coordinating the integrity of the biofilm through mediat-
ing microcolony formation and restraining the biovolume.
Our results indicate that all of these interactions are likely to
be coordinately essential for the initiation and development
of appropriately structured biofilms. To our knowledge, this
is the first report to evaluate the roles of long/short fimbriae
as well as gingipains on P. gingivalis biofilm formation.


2.0E+05 4.0E+05 6
Exopolysaccharide biovolume (pm





:-


0 0.2 0.4
Exopolysaccharide/cell ratio


Interestingly, the distinct fimbria types functioned differ-
ently in regard to biofilm formation. Our findings agree
with a recent report [17], which suggested that long fim-
.0E+05 briae are required for initial attachment and organization
3) ofbiofilms. In that study, it was also shown that short fim-
briae promoted bacterial autoaggregation, whereas long
fimbriae suppressed it. Other studies have shown that
-* autoaggregation is attributable to long fimbriae on the cell
surface [18,31,32], and deletion of short fimbriae
enhances autoaggregation [18], more consistent with our
present findings. However, it would appear that autoag-
gregation is context and assay dependent, and in any
0.6 event not a good predictor of accumulation on abiotic sur-
faces.


Figure 5
Exopolysaccharide production by P. gingivalis wild-
type strain and mutants in dTSB. A) Visualization of
exopolysaccharide production in biofilms formed by P. gingi-
valis strains after staining with FITC-labelled concanavalin A
and wheat germ agglutinin (green). Bacteria were stained
with DAPI (blue). Fluorescent images were obtained using a
CLSM. The z stack of the x-y sections was converted to com-
posite images with the "Volume" function using Imaris soft-
ware, after which a y stack of the x-z sections was created
and is presented here. B) Fluorescent images were quantified
using Imaris software and average of total exopolysaccharide
biovolume per field was calculated. C) Exopolysaccharide lev-
els are expressed as the ratio of exopolysaccharide/cells
(FITC/DAPI) fluorescence. The experiment was repeated
independently three times. Data are presented as averages of
8 fields per sample with standard errors of the means. Statis-
tical analysis was performed using a Scheffe test. *p < 0.05
and **p < 0.01 in comparison to the wild-type strain.



In the present study, the roles of long/short fimbriae and gin-
gipains on the initiation and development of biofilms
formed by P. gingivalis were examined. Interestingly, those
molecules were found to play distinct roles in the above-


Recently, it was reported that ClpXP, a proteolytic core
and associated ATPase unit of the bacterial stress response
system, negatively regulated the surface exposure of short
fimbriae, and a ClpXP mutant showed elevated monospe-
cies biofilm formation [33]. As we have shown here that
the short fimbria mutant MPG67 developed greater bio-
film accumulation than the wild type, it is likely that
ClpXP has numerous effects on cell surface molecules
important in biofilm development.

The long/short fimbriae mutant MPG4167 and RgpA/B
mutant KDP133 developed biofilms with significantly
large amounts of bacterial cells. In addition, the exopoly-
saccharide/cell ratio was significantly smaller than the
other strains, and the biofilms of these strains were shown
to be fragile (Figures 5C and 6). Rgp is an enzyme that
processes precursor proteins of bacterial surface compo-
nents such as fimbriae [22,23], therefore, Rgp-null
mutants exhibit defective surface protein presentation.
Thus not only MPG4167 but also KDP133 do not have
intact fimbrial protein on the cell surface, which might be
related to imperfect anchoring of exopolysaccharide on
the bacterial surfaces. The gingipains null mutant KDP 136
did not show the same tendency in spite of the lack of


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* Before sonic disruption
O After sonic disruption


Figure 6
Tenacity of biofilms formed by P. gingivalis wild tstrain and mutants. Standardized cultures of P. gingivalis were inocu-
lated into dTSB in saliva-coated 12-well polystyrene plate and incubated in a static manner at 37C for 60 hours, with the
resulting biofilms sonicated for I second. Immediately after sonication, supernatants containing floating cells were removed by
aspiration and the biofilm remains were gently washed with PBS. P. gingivalis genomic DNA was isolated from the biofilms and
the numbers of P. gingivalis cells were determined using real-time PCR. Relative amounts of bacterial cell numbers were calcu-
lated based on the number of wild-type cells without sonication considered to be 1.0. Percentages shown indicate the amount
of remaining biofilm after sonic disruption. The experiment was repeated independently three times with each strain in dupli-
cate. Standard error bars are shown. Statistical analysis was performed using a Scheffe test. *p < 0.05 and **p < 0.01 in compar-
ison to the wild-type strain.


both types of fimbriae, suggesting the presence of Kgp was
related to the unusual exopolysaccharide accumulation.
In contrast, long fimbriae mutant KDP150 formed a
tough and cohesive biofilm, and its exopolysaccharide/
cell ratio was significantly higher than the other strains.
Together, these findings suggest that the exopolysaccha-
ride/cell ratio seems to be related to the physical strength
of P. gingivalis biofilms.

The specific role of Kgp may involve regulation of biofilm
formation by the dispersion, de-concentration, and/or
detachment of microcolonies. Rgp also seemed to coordi-
nate the integrity of the biofilm in the developing phase as
well as maturation phase. There are several reports which
suggest that the present morphological changes in proteinase
mutants were possibly due to loss of proteolytic activities. In
Staphylococcus aureus, increased levels of serine proteases were
detected in detaching biofilm effluents, and a serine protease
inhibitor suppressed the biofilm detachment [34]. In the
same report, a double mutant in a metalloprotease and ser-
ine proteases, which displayed minimal extracellular pro-


tease activity, showed significantly enhanced biofilm
formation and a strongly attenuated detachment phenotype.
In Streptococcus pneumoniae, trypsin or proteinase K was
shown to inhibit biofilm development, and incubation of
mature biofilms with proteinase K drastically diminished the
number of biofilm-associated sessile cells [35]. Since our
data also showed that the mutation in gingipain genes
resulted in enhanced biofilm formation as well as a strongly
attenuated detachment phenotype, this suggests that protei-
nase domains of Kgp and Rgp are significantly involved in
biofilm regulation [5]. In addition, the tyrosine phosphatase
Ltpl reportedly dephosphorylated gingipains, resulting in
suppression of biofilm formation [10], which also supports
the involvement of gingipains as shown in this study. Fur-
thermore, the present gingipain mutants lacked proteinase
domains as well as C-terminal flanking segments coding for
hemaglutinin/adhesin (HA) domains [36]. Higher concen-
trations of iron in the cultivation media can have a positive
effect on the stability of the biofilms [37], thus decreased
hemin uptake due to the lack of HA domains might modu-
late the biofilm structures in dTSB.


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Table 4: P. gingivalis strains used in this study


Relevant properties


Reference


33277
KDP150
MPG67
MPG4167
KDP 129
KDP133
KDP136


Wild type
fimA::erm
mfa I ::erm
fimA::erm mfa I ::tetQ
kgp::cat
rgpA::tetQ rgpB::erm
rgpA::erm rgpB::tetQ kgp::cat


Autoaggregation driven by nonspecific hydrophobic
mechanisms is thought to contribute to hetero- and
homo-typic biofilm formation [24]. Indeed, the signifi-
cant change of autoaggregation efficiencies in KDP129,
KDP150, MPG67 and MPG4167 were found to be posi-
tively associated with alteration of biofilm structures
under the non-proliferation condition. However, such an
association was not observed in Rgp-null mutant strains,
KDP133 and KDP136, and was not significant under the
proliferation condition. Our present results suggested that
a biofilm-regulatory molecule Rgp does not function
through autoaggregation but rather through other mecha-
nisms mediating intimate contact among P. gingivalis
cells. Recently Kato et al. found that autoaggregation abil-
ity correlated poorly with the hydrophobicity in FimA-
substituted mutants [38]. In addition, the hydrophobicity
was reported not to depend on the presence or absence of
FimA on the bacterial surface [39]. In-depth mathematical
and physical examinations may be needed to explain the
complicated roles of hydrophobicity, autoaggregation
and cell surface structure on biofilm development.

Besides fimbria and proteinases, our findings indicate that
other molecules of P. gingivalis, which are not processed
by gingipains, mediate homotypic biofilm formation.
Indeed several factors, including a putative glycosyltrans-
ferase (PG_0106), UDP-galactose 4-epimerase (GalE),
internalin J protein (InlJ), a universal stress protein
(UpsA), and a low molecular weight tyrosine phosphatase
(Ltpl), have been reported to be required for homotypic
biofilm formation by P. gingivalis [10,19,40-42]. Autoin-
ducer-2, which regulates proteinase and hemagglutinin
activities, hemin and iron acquisition pathways, and stress
gene expression, is also considered to be involved in
homotypic biofilm formation [43-46]. It is possible that
these molecules also have effects in regard to biofilm
structure alterations, in addition to fimbriae and gingi-
pains. Further work is necessary to understand the com-
plete process of the biofilm formation by P. gingivalis.

Conclusion
The present results suggest distinct roles of long/short fim-
briae and gingipains in homotypic biofilm development


Wild type
Long fimbria (FimA)- deficient
Short fimbria (Mfal)- deficient
Long and short fimbria-deficient
Kgp-null
Rgp-null
Rgp/Kgp-null


ATCC
[20]
[18]
[18]
[20]
[20]
[20]


by P. gingivalis. Long fimbriae are initial positive media-
tors of biofilm formation, and thereafter they decrease the
expression of exopolysaccharide to regulate adhesive
properties. Short fimbriae as well as Kgp are negative reg-
ulators of microcolony formation. Rgp plays a bifunc-
tional role to coordinate the integrity of the biofilm
through mediating microcolony formation and restrain-
ing biovolume. Collectively, these molecules seem to act
coordinately to regulate the development of mature bio-
films.

Methods
Bacterial strains and media
The P. gingivalis strains used in this study are shown in
Table 4. P. gingivalis cells were inoculated from blood agar
plates and grown anaerobically (85% N2, 10% H2, 5%
CO2) at 37C in trypticase soy broth supplemented with
1 mg/ml of yeast extract, 1 pg/ml of menadione and 5 pg/
ml of hemin (TSB). At stationary phase, the cells were har-
vested by centrifugation at 6,000 x g for 7 minutes, resus-
pended in pre-reduced 10 mM phosphate buffer
containing 0.15 M sodium chloride (PBS; pH 7.4) and
then used in the assays. When necessary, the following
antibiotics were used at the concentrations shown in
parentheses: chloramphenicol (20 [tg/ml), erythromycin
(10 ag/ml), and tetracycline (1 ag/ml). To observe initial
attachment and organization of biofilms, P. gingivalis cells
were anaerobically incubated in pre-reduced PBS without
a nutrition source [ 19]. In order to monitor an increase in
biovolume due to cell division as well as exopolysaccha-
ride accumulation, bacterial cells were cultured in TSB
medium diluted with PBS (dTSB; TSB/PBS ratio, 1:2) [47].

Autoaggregation assay
An autoaggregation assay was essentially performed as
described previously [481. Briefly, 1 ml of P. gingivalis sus-
pension (4 x 108 cells) was transferred into a UV-cuvette
then incubated at 37 C with stirring. Autoaggregation was
monitored by measuring the decrease in optical density at
A550 (OD550) using a UV-visible recording spectropho-
tometer (UV-265FW; Shimadzu Co. Kyoto, Japan). Dur-
ing the incubation, dA/dt was continuously calculated
and recorded by subtraction of At, the absorbance at time


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Strain


Genotype


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t min, from At+, at time (t + 1) min. The maximum value
of dA/dt in this curve was used as the autoaggregation
activity [48]. The data represent the mean + standard error
of three separate experiments with each strain in dupli-
cate.

Saliva
Saliva stimulated by mastication of paraffin balls was col-
lected in a sterile centrifuge tube on ice from healthy
donors and pooled, as described previously [49]. Dithio-
threitol (Sigma-Aldrich, St. Louis, MO) was added to a 2.5
mM final concentration, then the saliva was gently stirred
on ice for 10 minutes and centrifuged at 3,000 x g for 20
minutes at 4C. The clarified saliva supernatant was
decanted, 3 volumes of distilled water was added, and the
25% saliva was filtered through a 0.20 upm pore size filter
and frozen in 40 ml aliquots. Immediately prior to use,
the sterile saliva was thawed at 37 o C; the slight precipitate
was pelleted at 1,430 x g for 5 min, and the clear 25%
saliva supernatant was used in experiments.

Microscope observation
Quantitative and structural analysis of homotypic P. gingiva-
lis biofilms was accomplished by confocal laser scanning
microscopy (CLSM, Radiance 2100, Bio-Rad) and subse-
quent image analysis [50]. P. gingivalis was stained with CFSE
(8 pg/ml; Molecular Probes, Eugene, OR), washed three
times and 1 x 108 cells in PBS or dTSB were anaerobically
incubated in a 25% saliva-coated wells of a chambered cov-
erglass system (Culture Well', Grace Bio Labs, Bend, OR) for
24 hours at 37 C in the dark on a rotator. The resulting bio-
films were examined using the CLSM with reflected laser
light at 488 nm. The images were analyzed using the Image I
1.34s (National Institutes of Health; Bethesda, MD) and
Imaris 5.0.1 (Bitplane AG; Zurich, Switzerland) software
packages. The experiment was repeated independently three
times with each strain in triplicate.

Biofilm characterization by image analysis
Z stacks of the x-y sections in the CLSM images were con-
verted to composite images with the "Iso Surface" func-
tion of the "Surpass" option provided by Imaris 5.0.1
(Bitplane AG; Zurich, Switzerland) software. Iso Surface
images were created at a threshold of 40 and smoothed
with the "Gaussian Filter" function at a width of 1.28 [pm,
then the biovolume was calculated. Measurement of peak
parameters was performed as described previously [50].
Digitally reconstructed images of the x-z section, 189.4 Pmn
x appropriate height with 10-pm spaced y-series slices,
were created using the "Reslice" function of Image J. An
image series of the x-z section was processed using the
"Find Edges" function, then the peak height was calcu-
lated by Image J. Color images of the x-z section were con-
verted into gray scale and the density per vertical position
(x-axis) was analyzed with the "Plot profile" function of


Image J. The data were then exported as plot values with
x-axis distance information. Peaks were defined as posi-
tions where plot values were higher than on either side,
and the distance between two peaks was measured. The
peak number was counted in a 90-pm section of the x-axis.

Exopolysaccharide production assay
P. gingivalis organisms were stained with DAPI (50 [tg/ml;
Molecular Probes, Eugene, OR), then washed and cul-
tured in 25% saliva-coated wells of CultureWell cham-
bered coverglass system with dTSB for 24 hours. The
resulting biofilms were washed, then exopolysaccharide
was labelled with Concanavalin A-FITC and Wheat germ
agglutinin-FITC (100 pg/ml; Molecular Probes) for 30
minutes at room temperature, as described previously
[10]. After washing, fluorescent images were obtained
using CLSM with reflected laser light at 405 and 488 nm,
then analyzed as described above. The images were
obtained with 8 fields per a sample. The experiment was
repeated independently three times.

Sonic disruption assay
A 12-well polystyrene plate (#1820-024, AGC Techno
Glass, Chiba, Japan) was coated with 25% saliva. P. gingi-
valis cells (4 x 108 cfu/well) were incubated in a static
manner in dTSB for 60 hours at 370C and the resulting
biofilms were sonicated for 1 second at output level 1
(output power: 25 W, oscillating frequency: 28 kHz, tip
diameter: 2.5 mm) with a Handy ultrasonic disruptor
(UR-20P, Tomy Seiko, Tokyo, Japan). During sonication,
the oscillator was fixed with a stand, and the tip of horn
was positioned 5 mm above from the center point of flat
well bottoms. Immediately after the sonication, supema-
tants containing floating cells were removed by aspiration
and the remaining biofilms were gently washed with PBS.
P. gingivalis genomic DNA was isolated from the biofilms
and the number of P. gingivalis cells per well was deter-
mined using real-time PCR, as described previously [51].
The data represent the means + standard error of three
separate experiments with each strain in duplicate.

Statistical analyses
All data are expressed as the mean + standard error. Mul-
tiple comparisons were performed by one-way analysis of
variance and Sheffe's test using the SPSS 16.01 software
(SPSS Japan Inc., Tokyo).

Abbreviations
TSB: trypticase soy broth supplemented with 1 mg/ml of
yeast extract, 1 [tg/ml of menadione and 5 [tg/ml of
hemin; DAPI: 4',6-diamino-2-phenylindole; FITC: fluo-
rescein isothiocyanate; CFSE: 5-(and-6)-carboxyfluores-
cein succinimidyl ester; CLSM: confocal laser scanning
microscopy; dTSB: diluted TSB medium.



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BMC Microbiology 2009, 9:105


Authors' contributions
MK carried out the microscope observation, image analy-
sis and autoaggregation assay, as well as prepared the ini-
tial draft of the manuscript. AA conceived of the study and
helped to draft the manuscript. EH and YY carried out the
sonic disruption assay. HI performed the statistical analy-
sis. KN and NH provided P. gingivalis knockout mutants
used in this study. GDT developed the exopolysaccharide
assay for P. gingivlais. RJL participated in the design of the
study and helped to draft the manuscript. SS participated
in the design of the study and coordination. All authors
read and approved the final manuscript.


Acknowledgements
This research was supported in part by a grant from the 21st Century
Center of Excellence program entitled "Origination of Frontier BioDen-
tistry" held at Osaka University Graduate School of Dentistry, as well as
grants-in-aid for Scientific Research on Priority Areas and grants-in-aid for
Scientific Research from the Ministry of Education, Culture, Sports, Science
and Technology, and DEI2505 from the NIH

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