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Title

Fusobacterium nucleatum enhances invasion of human

gingival epithelial and aortic endothelial cells by

Porphyromonas gingivalis

Author(s)

Alternative

Saito, A; Inagaki, S; Kimizuka, R; Okuda, K;

Hosaka, Y; Nakagawa, T; Ishihara, K

JournalFEMS immunology and medical microbiology, 54(3):

349-355

URL http://hdl.handle.net/10130/1121

RightThe definitive version is available at

www.blackwell-synergy.com

1

Research Article

REVISED

Fusobacterium nucleatum enhances invasion of human gingival

epithelial and aortic endothelial cells by Porphyromonas gingivalis

Atsushi Saito1,2, Satoru Inagaki1,3, Ryuta Kimizuka1,3, Katsuji Okuda3, Yasuo

Hosaka4, Taneaki Nakagawa4 & Kazuyuki Ishihara1, 3

1Oral Health Science Center, Tokyo Dental College, Chiba, Japan, 2Department

of Clinical Oral Health Science, Tokyo Dental College, Tokyo, Japan,

3Department of Microbiology, Tokyo Dental College, Chiba, Japan, 4Department

of Dentistry and Oral Surgery, Faculty of Medicine, Keio University, Tokyo, Japan

Keywords:

Bacterial invasion; Porphyromonas gingivalis; Fusobacterium nucleatum;

endothelial; gingival epithelial; polymicrobial infection

Short running title:

Polymicrobial infection of endothelial cells

Correspondence: Atsushi Saito, DDS, PhD

Department of Clinical Oral Health Science, Tokyo Dental College.

2-9-18 Misaki-cho, Chiyoda-ku, Tokyo 101-0061, Japan

Tel: +81 3 5275 1721; fax: +81 3 3262 3420; e-mail: atsaito@tdc.ac.jp

2

Abstract

Invasion by Porphyromonas gingivalis has been proposed as a possible mechanism of

pathogenesis in periodontal and cardiovascular diseases. P. gingivalis have direct access

to the systemic circulation and endothelium in periodontitis patients by transient

bacteremia. Periodontitis can be described as one of the predominant polymicrobial

infections of humans. In the present study, P. gingivalis strains were tested for their

ability to invade a human gingival epithelial cell line (Ca9-22) and human aortic

endothelial cells (HAEC) in co-infection with Fusobacterium nucleatum using

antibiotic protection assays. Co-infection with F. nucleatum resulted in 2- to 20- fold

increase in invasion of host cells by P. gingivalis strains. The invasive abilities of P.

gingivalis strains were significantly greater when incubated with a F. nucleatum clinical

isolate (which possesses strong biofilm-forming ability), than when incubated with F.

nucleatum type strain. In inhibition assays with metabolic inhibitors, difference in

inhibition profiles was noted between mono- and polymicrobial infections. Collectively,

our results suggest that F. nucleatum facilitates invasion of host cells by P. gingivalis.

Investigations of polymicrobial infection of host cells should improve our

understanding of the role of P. gingivalis in periodontal infection and proatherogenic

mechanisms.

3

Introduction

Epidemiological studies have demonstrated a positive association between periodontitis

and cardiovascular diseases (Beck et al., 1996). It has been suggested that one of the

linkage between these diseases is directly infectious, i.e., bacteria within the atheroma

may be involved in the development of the atherosclerotic plaque (Chiu, 1999; Lalla et

al., 2003; Gibson III et al., 2004). Periodontal pathogens including Porphyromonas

gingivalis have been detected in atherosclerotic plaques in humans using PCR

techniques (Haraszthy et al., 2000; Ishihara et al., 2004, Kozarov et al., 2006).

P. gingivalis elicits a proatherogenic response in endothelial cells in the form of

increased leukocyte adhesion with concomitant up-regulation of adhesion molecules,

heightened production of proinflammatory cytokines and chemokines, as well as an

induction of prothrombotic properties (Kang & Kuramitsu, 2002; Roth et al., 2007).

Interestingly, these effects on endothelial cells cannot be attributed to a sole effect of

stimulation by bacterial cell-surface components, but may require the invasion of host

cells by viable bacteria (Darveau et al., 2002; Roth et al., 2007).

Invasion by P. gingivalis has been proposed as a possible mechanism of pathogenesis

in periodontal and cardiovascular diseases (Lamont et al., 1995; Deshpande et al., 1998).

P. gingivalis has direct access to the systemic circulation and the endothelium in

periodontitis patients, as transient bacteremias are common (Kinane et al., 2005), and

the ability of P. gingivalis, detected at the sites of atherosclerotic disease, to invade host

cells has been demonstrated (Kozarov et al., 2005).

Periodontitis can be described as one of the predominant polymicrobial infections of

humans (Brogden et al., 2005). Since periodontal diseases result from complex

interactions of multiple microorganisms, it is essential to investigate interactions

between different periodontal bacteria and host cells. Bacterial species in subgingival

4

plaque have been shown to fall into five distinctive complexes of closely related species

(Socransky et al., 1998). Bacteria in one of the complexes referred to as “red complex”

are commonly associated with periodontal lesions. However, bacteria in this complex

such as P. gingivalis are usually detected in the presence of bacteria from a closely

related complex referred to as “orange complex” comprising e.g. Fusobacterium

nucleatum (Socransky et al., 1998, Kesavalu et al., 2007). Antagonistic and synergistic

physiologic mechanisms, as well as environmental selection are thought to be involved

in such relationships (Kesavalu et al., 2007). F. nucleatum initially adheres to early

colonizers, including gram-positive cocci, and enhances the adherence of other

periodontopathic bacteria including P. gingivalis (Kolenbrander, 2000).

In polymicrobial infections by bacterial enteropathogens, it has been shown that the

ability of Campylobacter jejuni to invade cultured epithelial cells is significantly

enhanced by the presence of other enteropathogens as coinfectants (Bukholm &

Kapperud, 1987). Whether similar interaction occurs in periodontopathogens is

unknown. Data on the potential of P. gingivalis invasion into host cells in polymicrobial

infection are scarce; the present study therefore sought to investigate the capacity of P.

gingivalis to invade human gingival epithelial and aortic endothelial cells in

co-infection with F. nucleatum.

5

Materials and Methods

Bacterial strains and growth conditions

P. gingivalis ATCC 33277, P. gingivalis W83 (ATCC BAA-308), F. nucleatum TDC100

[a clinical isolate and working strain in our laboratory (Saito et al., 2008) ] and F.

nucleatum ATCC 25586 were routinely maintained on tryptic soy agar (Difco

Laboratories, Detroit, MI) supplemented with 10% defibrinated horse blood, hemin (5

µg mL-1) and menadione (0.5 µg mL-1) at 37 °C under anaerobic conditions.

Escherichia coli SCS110 and DH5α strains were used as a control in antibiotic

protection assays.

Cells and culture conditions

Human gingival epithelial cell line, Ca9-22, was purchased from Health Science

Research Resources Bank (Osaka, Japan). Ca9-22 is an established transformed human

gingival cell line, which has been used in previous studies as a culture model of oral

epithelial cells (Hirose et al., 1999; Ohshima et al., 2001; Takeuchi et al., 2008). The

Ca9-22 cells were maintained in Eagle’s minimal essential medium (MEM)

supplemented with glutamine (0.6 g L-1), heat-inactivated 10 % fetal calf serum, and

gentamicin (50µg mL-1) / amphotericin B (50ng mL-1) (Cascade Biologics, Portland,

OR) at 37 °C in 5 % CO2.

Human aorta endothelial cells (HAEC) were supplied by Kurabo Inc. (Osaka, Japan)

and maintained in HuMedia-EG2 (Kurabo) under an atmosphere of 5% CO2 and 95 %

air at 37 °C. Cells from passages 4 through 9 were tested for viability and morphology

prior to seeding in appropriate tissue culture plates and allowed to reach

near-confluency before assay.

Invasion procedures

Invasion of bacteria was quantitated by the standard antibiotic protection assay (Lamont

6

et al, 1995, Deshpande et al., 1998). Briefly, epithelial cells were seeded in 12-well

flat-bottom culture plate (Iwaki, Chiba, Japan) at a cell density of 2.0 x 105 cells per

well. Prior to infection, the cells were washed twice with phosphate-buffered saline

(PBS, pH 7.4) and incubated further 2 h in MEM without antibiotics. The multiplicity of

infection (MOI) was calculated based on the number of cells per well at confluence. P.

gingivalis and F. nucleatum strains were inoculated into brain heart infusion broth

(Becton Dickinson, Sparks, MD) supplemented with 0.5 % of yeast extract, hemin (5 µg

mL-1) and menadione (0.5µg mL-1), and grown for 2 days until the optical density at 660

nm reached 1.0. After washing with PBS, bacterial cells were resuspended in MEM.

Bacterial suspensions (2.0 x 107 cells per well) were added to confluent Ca9-22

monolayers (MOI=100) and incubated at 37°C in 5 % CO2 for 2h. After incubation,

unattached bacteria were removed following washing of the monolayers 3 times with

PBS. External adherent cells were then killed by incubating the infected monolayers

with MEM containing 200 µg mL-1 of metronizazole and 300 µg mL-1 of gentamicin for

1 h. This concentration of antibiotic was sufficient to completely kill 108 bacteria per

mL in 1h. Controls for antibiotic killing of bacteria were included in all experiments.

After exposure to antibiotic, monolayers were washed twice with PBS, and lysed in 1

mL of sterile distilled water per well. Cells were incubated aerobically for 30 min,

during which they were disrupted by repeated pipetting. Lysates were diluted and plated

on blood agar plates, and incubated anaerobically at 37°C for 10 days. CFU of invasive

organisms were then enumerated. Invasion efficiency was expressed as the percentage

of the initial inoculum recovered after antibiotic treatment and Ca9-22 lysis.

The invasion assay with HAEC was performed using the same procedure as above

with EG2 medium.

7

Polymicrobial infection

Following demonstration of monomicrobial infections with P.gingivalis strains, we

performed experiments to develop a model of polymicrobial periodontal infection using

P. gingivalis and F. nucleatum as members of a prototype consortium, and examined the

invasion characteristics and interactions of these organisms. For polymicrobial infection,

P. gingivalis ( 1 x 107 cells mL-1) was gently mixed with an equal volume of F.

nucleatum ( 1 x 107 cells mL-1) and the organisms were allowed to interact for 5 min.

For the monomicrobial control infection, P. gingivalis was mixed with an equal volume

of MEM or EG2 medium. As a control for polymicribial infection, E. coli SCS110 or

DH5α was pre-incubated with P. gingivalis. The poly- and monomicrobial inocula were

added to Ca9-22 or HAEC monolayers.

The bacterial culture growth phase, viability, counts, interaction times, suspension

medium, infection dose, and infection procedures were all standardized; i.e., the same

preparation and infection protocols were used for all invasion assays throughout the

study.

Inhibition of bacterial invasion

For inhibition assays with antiserum, P. gingivalis or F. nucleatum cells were

preincubated with the indicated dilution of rabbit polyclonal anti-P.gingivalis serum for

30 min at RT prior to use in assays.

P. gingivalis or F. nucleatum cells were also preincubated with indicated

concentrations of D-galactose (inhibitor of F. nucleatum adhesion/invasion) for 15 min

at RT prior to use in assays.

To dissect the biochemical pathways involved, the effect of a group of metabolic

inhibitors on invasion was investigated. The following inhibitors, in the solvent and at

the final concentration indicated, were used. Cytochalasin D, 1 µg mL-1 in dimethyl

8

sulfoxide (DMSO); nococazole, 10 µg mL-1 in DMSO; staurosporine, 0.5 µM in

DMSO; cycloheximide, 100 µg mL-1 in ethanol. The inhibitors were preincubated with

the host cells for 60 min prior to addition of the bacteria and remained present

throughout the invasion assay. All potential inhibitors were tested at the concentration

used for possible adverse effects on the host cells, through comparison to cells without

inhibitors, by examining the morphology of the cells and the confluency of the

monolayer.

Data and statistical analysis

All experiments were performed in duplicate or triplicate for each condition and

repeated at least three times. Statistical comparisons were performed using a software

package (InStat 3.0, GraphPad Software Inc., La Jolla, CA). Mann-Whitney U test or

analysis of variance (ANOVA) with Bonferroni post test (for multiple comparisons)

was used.

9

Results

Monomicrobial infection

There was strain to strain variability in the ability of P. gingivalis to invade HAEC and

Ca9-22 (Fig. 1). For both HAEC and Ca9-22, P. gingivalis strain 33277 had greater

invasive abilities than strain W83. For HAEC, mean invasion efficiencies for P.

gingivalis 33277 and W83 were 1.58 % and 0.52 %, respectively (Fig. 1a). The ability

of P. gingivalis 33277 to invade Ca9-22 was essentially equal to its ability to invade

HAEC (Fig. 1b); however, P. gingivalis W83 invaded HAEC approximately 3-fold over

Ca9-22.

Polymicrobial infection with P.gingivalis and F.nucleatum

In polymicrobial infection experiments with P. gingivalis and F. nucleatum, viable

counts of P. gingivalis strains recovered from HAEC or Ca9-22 cells were used to

determine bacterial invasion. Co-incubation of P. gingivalis 33277 with F. nucleatum

significantly boosted P. gingivalis invasion of host cells, resulting in 2- to 20- fold

increase in invasion efficiencies (Fig.1). Co-incubation with E. coli SCS110 or DH5α

exerted no effect on P. gingivalis invasion of HAEC or Ca9-22.

Invasion of host cells by P. gingivalis 33277 was statistically significantly higher in

the presence of F. nucleatum TDC100 (invasion efficiency: 12 %) than F. nucleatum

25586 ( 6% ) (P < 0.01)(Fig. 1a). In the presence of F. nucleatum, P. gingivalis W83

which showed low invasive ability in monomicrobial infection, demonstrated dramatic

increase in invasion. Invasion of HAEC with P. gingivalis W83 was significantly more

enhanced in the presence of F. nucleatum TDC100 (invasion efficiency: 8 %) than F.

nucleatum 25586 ( 2% ) (P < 0.01). Similarly, F. nucleatum significantly enhanced

invasion of P. gingivalis strains to Ca9-22 cells (Fig. 1b).

10

Inhibition of bacterial invasion

P. gingivalis invasion of HAEC was inhibited by anti-P. gingivalis serum (diluted 1:100)

by approximately 70 % (Table 1). The inhibition was significant, but relatively low

when co-incubated with F. nucleatum. A similar trend was observed with Ca9-22 cells

(data not shown).

To determine whether the enhanced invasion of P. gingivalis in polymicrobial

infection involves a lectin-like adhesin(s), sugar inhibition assay was performed.

Incubation with D-galactose resulted in decreased invasion by P. gingivalis in

polymicrobial infection experiments (Fig.2).

Various metabolic inhibitors previously reported to reduce P. gingivalis or F.

nucleatum invasion were assessed for the ability to inhibit Fusobacterium-enhanced P.

gingivalis invasion. In mono- and polymicrobial infection experiments, invasion of the

host cells by P. gingivalis required multiple components of the host including actin,

microtubule, and protein kinases (Table 2). One notable difference in inhibition profiles

was observed between mono- and polymicrobial infections. Cycloheximide (which

target host cell protein synthesis) significantly reduced invasion by P. gingivalis in

polymicrobial infection experiments. This inhibitor has previously been shown to

inhibit F. nucleatum invasion but not P. gingivalis invasion.

11

Discussion

In the present study, we first explored the abilities of different P. gingivalis strains to

invade gingival epithelial and endothelial cells. P. gingivalis W83 has been shown to be

highly virulent in experimental animal models (Neiders et al., 1989, Genco et al., 1991).

In our experimental set-up, invasive ability of P. gingivalis W83 into host cells was

relatively low when compared to P. gingivalis 33277, which has been shown to be

highly fimbriated but less virulent. Furthermore, P. gingivalis W83 displayed an

invasive ability that differed in the tested cell types. Fimbriae are considered important

in adherence and invasion by P. gingivalis. However, it has been shown that the

presence and expression of fimA is not sufficient for P. gingivalis invasion of endothelial

and epithelial cells( Dorn et al., 2000; Umeda et al., 2006). The differential invasion

efficiency observed for different cell types is likely due to different interactions between

P. gingivalis and the types of cell surface receptors present on the different cell types

that are involved in the invasion process.

There is increasing evidence in the literature for the importance of polymicrobial

infections in which selected microorganisms interact in a synergistic or antagonistic

fashion, impacting on pathogenesis of periodontal disease (Chen et al., 1996; Feuille

et al., 1996; Kesavalu et al., 2007). Synergistic interactions in virulence between F.

nucleatum and P. gingivalis have been observed in vitro and in animal models (Feuille

et al, 1996; Ebersole et al., 1997). In this study, we demonstrated that F. nucleatum

enhances the ability of P. gingivalis to invade host cells. To the best of our knowledge,

this study is the first to demonstrate such an interaction between F. nucleatum and P.

gingivalis.

In polymicrobial infection experiments, F. nucleatum TDC 100 enhanced P .

gingivalis invasion of host cells significantly more than F. nucleatum type strain. A

12

previous study from our group has demonstrated that F. nucleatum TDC 100 has a

synergistic relationship with P. gingivalis and a strong biofilm forming ability (Saito et

al., 2008). Han et al. (2000) reported that a spontaneous lam mutant F. nucleatum,

defective in aggregation with human lymphocytes and coaggregation with P. gingivalis,

was defective in attachment to and invasion of human gingival epithelial cells,

suggesting that same bacterial determinants are involved in aggregation properties and

ability to invade host cells. F. nucleatum and P. gingivalis are strong coaggregating pairs,

and the coaggregation may have the capacity to alter the expression of virulence factors

in individual microorganisms (Feuille et al., 1996). Coaggregation between P. gingivalis

and F. nucleatum is mediated by a galactoside moiety on the P. gingivalis surface and a

lectin on the F. nucleatum, and inhibited by lactose, galactose and related

monosaccharides (Kolenbrander & Anderson, 1989; Kinder & Holt, 1993). We have

observed a strong coaggregation reaction between P. gingivalis 33277 and F. nucleatum

TDC 100, that is inhibitable by galactose (data not shown). The synergistic interactions

between F. nucleatum and P. gingivalis observed in the present study could be partly

explained by coaggregating effect between these organisms, as galactose also inhibited

F. nucleatum enhanced P. gingivalis invasion.

In the present study, co-infection with F. nucleatum strains markedly enhanced

invasion of host cells by P. gingivalis W83, a strain we have shown to be minimally

invasive in monomicrobial infection of host cells. Rudney et al. (2005) showed that

intracellular infections of buccal epithelial cells with periodontal pathogens were

uniformly polymicrobial, and proposed several scenarios regarding invasion of host

cells by a consortium of oral bacteria. Invasiveness might be limited to a subset of oral

species that use it as a virulence factor. Alternatively, a wide range of oral bacteria

which principally live in biofilm might be capable of invasion as a means of persisting.

13

Since species interaction appears to be widespread in oral biofilm (Cook et al., 1998;

Palmer et al., 2001; McNab et al, 2003), another alternative could be that non-invasive

species gain entrance to cells by forming consortia with invasive species. It has been

reported that F. nucleatum transports noninvasive Streptococcus cristatus into human

epithelial cells (Edwards et al., 2006). In the present study, polymicrobial infection of

the host cells by P. gingivalis and F. nucleatum not only facilitated P. gingivalis invasion,

but also resulted in the invasion by F. nucleatum, although the extent of F. nucleatum

invasion was relatively low, when compared to that of P. gingivalis 33277 (data not

shown). Although anti-P. gingivalis serum abrogated P. gingivalis invasion of host cells

in a monomicrobial infection setting, the extent of inhibition was less in polymicrobial

infection. These results suggested that mechanisms other than adherence signal induced

by P. gingivalis are likely to be involved, and that interaction(s) between F. nucleatum

and host cells may play a significant role in fusobacterium-enhanced P. gingivalis

invasion.

Also in the inhibition experiment, cycloheximide significantly reduced invasion by P.

gingivalis in polymicrobial infection. As this inhibitor has previously been shown to

inhibit F. nucleatum invasion (Han et al., 2000) but not P. gingivalis invasion

(Deshpande et al., 1998), it is conceivable that infection by F. nucleatum may pave

the way for increased invasion of P. gingivalis.

We cannot yet clarify whether the effect exerted by the coinfectant is directed at the

host cell or the P. gingivalis. Invasive bacteria generally gain entry by co-opting and

re-directing host cell mechanisms such as endocytosis (Lamont et al., 1995; Sandros et

al., 1996; Progulske-Fox et al., 1999; Meyer et al., 1999; Han et al., 2000).

Immunomodulating roles of F. nucleatum have been suggested by previous studies

(Feuille et al., 1996; Choi et al., 2001). Polymicrobial infections may actually modulate

14

the adaptive host responses, leading to more effective evasion of protective immune

responses.

In summary, this report demonstrates that F. nucleatum facilitates P. gingivalis

invasion of human gingival epithelial and endothelial cells. The significance of this

increased ability to invade cells in progression of periodontal as well as cardiovascular

diseases needs to be elucidated in future studies. We are currently investigating

molecular mechanisms involved in this relationship, and whether other

periodontopathogens in a consortium are able to induce the synergistic effects.

Acknowledgement

The authors thank David Blette for editing the manuscript.

This research was supported by Oral Health Science Center Grant HRC7 from Tokyo

Dental College, and by a “High-Tech Research Center” Project for Private Universities:

matching fund subsidy from MEXT (Ministry of Education, Culture, Sports, Science

and Technology) of Japan, 2006-2010.

15

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Figure legends

(a)

(b)

Fig. 1

Invasion of (a) HAEC (b) Ca9-22 by P. gingivalis 33277 or P. gingivalis W83 in

mono- or polymicrobial infection with F. nucleatum strains. (MOI=1:100, infected

for 2h). Invasion efficiency (%) was expressed as percentage of the inoculum (P.

gingivalis) protected from the antibiotic killing for 1h. Values are the means ±

standard deviations of triplicate independent determinations from a typical

experiment. *Statistically significantly different (P < 0.01, ANOVA with Bonferroni

multiple comparisons test).

21

Fig. 2

Inhibition of fusobacterial enhanced P. gingivalis invasion by D-galactose. The

effects of increasing concentrations of D-galactose on P. gingivalis invasion of

HAEC were assessed for monomicrobial (P. gingivalis only; open circles), or

polymicrobial (P. gingivalis and F. nucleatum together; filled squares) infections.

Values are the means ± standard deviations of triplicate independent

determinations from a typical experiment. *Statistically significantly different from

control (P < 0.01, Mann-Whitney U test).

22

Table 1. Effect of anti-P.gingivalis serum on P.gingivalis invasion of HAEC by

mono- or polymicrobial infection

Invasion of HAEC (% of control) a Pre-incubation

Mono-infection (Pg) b Poly-infection (Pg + Fn) c

Control (Pre-immune serum)

Anti-Pg serum (dilution 1:1000)

Anti-Pg serum (dilution 1: 100)

100 ± 12

79 ± 12*

29 ± 20*

100 ± 21

89 ± 18

58 ± 11*

a Invasion of P.gingivalis 33277 relative to the level obtained in the absence of

antiserum (pre-immune serum control). Values given as means ± standard

deviations of triplicate independent determinations from a typical experiment. b Cells were infected by P. gingivalis 33277 c Cells were infected by P. gingivalis 33277 and F. nucleatum TDC100

*Statistically significantly different from control (P < 0.01) by ANOVA with

Bonferroni multiple comparisons test

23

Table 2. Effects of metabolic inhibitors on P. gingivalis invasion of HAEC by

mono- or polymicrobial infection

Invasion of HAEC (% of untreated control) a Inhibitor Target

Mono-infection (Pg) b Poly-infection (Pg + Fn) c

Cytochalasin D

Nocodazole

Saturosporine

Cycloheximide

Actin

Microtubule

Protein kinase

Protein synthesis

11.5 ± 3.5*

14.8 ± 5.0*

54.4 ± 5.5*

91.7 ± 5.5

23.0 ± 11.1*

18.2 ± 16.4*

22.7 ± 17.9*, †

25.7 ± 8.3*, †

a Invasion of P.gingivalis 33277 relative to the level obtained in the absence of

inhibitor (medium control). Values given as means ± standard deviations of

triplicate independent determinations from a typical experiment. b Cells were infected by P. gingivalis 33277 c Cells were infected by P. gingivalis 33277 and F. nucleatum TDC100

*Statistically significantly different from control (P < 0.01) by ANOVA with

Bonferroni multiple comparisons test †Statistically significantly different from monomicrobial infection (P < 0.01) by

Mann-Whitney U test