# Desulfovibrio spp. survive within KB cells and modulate inflammatory responses

C Bisson-Boutelliez, F. Massin, D. Dumas, N Miller, A. Lozniewski

► To cite this version:

C Bisson-Boutelliez, F. Massin, D. Dumas, N Miller, A. Lozniewski. Desulfovibrio spp. survive within KB cells and modulate inflammatory responses. Molecular Oral Microbiology, 2010, 25 (3), pp.226-35. 10.1111/j.2041-1014.2009.00550.x. http://hal-02987410

HAL Id: hal-02987410

https://hal.univ-lorraine.fr/hal-02987410

Submitted on 3 Nov 2020

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
<figure><img src="image_2.png" /></figure>

# *Desulfovibrio* spp. survive within KB cells and modulate inflammatory responses

C. Bisson-Boutelliez<fnref n="1" />,<fnref n="2" />, F. Massin<fnref n="3" />, D. Dumas<fnref n="4" />, N. Miller<fnref n="2" /> and A. Lozniewski<fnref n="1" />

**Correspondence:** Neal Miller, Département de Parodontologie et d'Implantologie, Faculté d'Odontologie, 96 avenue de Lattres de Tassigny, 54004 Nancy Cedex, France Tel.: + 33 3 83 85 29 52; fax: + 33 3 83 68 29 81; E-mail: neal.miller@odonto.uhp-nancy.fr

**Keywords:** cytokines; epithelial cells; invasion; periodontal disease

Accepted 1 September 2009

## SUMMARY

*Desulfovibrio* are sulfate-reducing anaerobic gram-negative rods that have been proposed as potential periodontopathogens. We investigated the capacity of *Desulfovibrio* to invade epithelial cells and induce cytokine secretion from these cells. *Desulfovibrio* strains were co-cultured with KB cells and counts of intracellular bacteria evaluated up to 3 days after infection. *Desulfovibrio desulfuricans* and *Desulfovibrio fairfieldensis* were able to survive within epithelial cells. Intracytoplasmic location of both bacterial species was confirmed by confocal laser scanning microscopy and transmission electron microscopy. Invasion was sensitive to nocodazole, an inhibitor of microtubule polymerization, but not to cytochalasin D, a microfilament inhibitor, suggesting that microtubule rearrangements were involved in the internalization of *Desulfovibrio* strains by KB cells. Infection by *Desulfovibrio* resulted in increased production of IL-6 and IL-8 by KB cells. The ability of *D. desulfuricans* and *D. fairfieldensis* to survive within oral epithelial cells and to modulate the epithelial immune response may contribute to the initiation and progression of periodontal diseases.

## INTRODUCTION

Periodontitis is a multifactorial disease involving complex interactions between various anaerobic

bacteria and host cells, which may lead to periodontal tissue destruction. Epithelial cell invasion has been suggested to play an important role in the pathogenesis of periodontitis (Lamont & Yilmaz, 2002; Colombo et al., 2007; Johnson et al., 2008). Cell invasion may allow periodontopathogens to persist within oral epithelial cells, to evade the immune system, and possibly to disseminate through underlying tissues (Andrian et al., 2004). So far, invasion potential has been shown *in vitro* and/or *in vivo* for several bacteria associated with periodontal diseases, including *Aggregatibacter actinomycetemcomitans*, *Eikenella corrodens*, *Fusobacterium nucleatum*, *Prevotella intermedia*, *Porphyromonas gingivalis*, *'Tannerella forsythia'*, and *Treponema denticola* (Meyer et al., 1991; Duncan et al., 1993; Dorn et al., 1998; Han et al., 2000; Colombo et al., 2007). While the interaction of all these pathogens with epithelial cells may contribute to the release of inflammatory mediators, it has been shown that the expression of various cytokines, including interleukin-1β (IL-1β), IL-6, and IL-8, by epithelial cells infected with *P. gingivalis* may be positively correlated with its invasive capacity (Eick et al., 2006). Consequently, epithelial invasion may also have a direct impact on disease progression and the inflammatory processes.

Sulfate-reducing bacteria (SRB) are a diverse group of organisms that are found in the environment

<footnote n="1">1 Laboratoire de Bactériologie-UMR CNRS 7565, Faculté de Médecine, Nancy Université, Nancy, France</footnote>

<footnote n="2">2 Département de Parodontologie et d'Implantologie, Faculté d'Odontologie, Nancy Université, Nancy, France</footnote>

<footnote n="3">3 Laboratoire d'Immunologie, Faculté de Médecine, Nancy Université, Nancy, France</footnote>

<footnote n="4">4 UMR CNRS 7563, Faculté de Médecine, Nancy Université, Nancy, France</footnote>
but may also colonize the digestive tract of animals
and humans (Gibson, 1990; Willis *et al.*, 1995; Zinke-
vich & Beech, 2000; Boopathy *et al.*, 2002). These
organisms have been reported to be the causes of
human infections including bacteremia and brain and
liver abscesses, and may play a role in inflammatory
bowel diseases (Watanabe *et al.*, 2007; Rowan
et al., 2009). Recent findings suggest that SRB may
also be involved in periodontitis (Langendijk *et al.*,
1999, 2000; Langendijk-Genevaux *et al.*, 2001;
Boopathy *et al.*, 2002; Loubinoux *et al.*, 2002; Vianna
et al., 2008). Different isolates of SRB have been
isolated from the oral cavity, generally belonging to
the genus *Desulfovibrio* (Langendijk *et al.*, 2001;
Boopathy *et al.*, 2002; Loubinoux *et al.*, 2002). So
far, *Desulfovibrio fairfieldensis* and, to a lesser
extent, *Desulfovibrio desulfuricans*, are the two
*Desulfovibrio* species that have been isolated from
the oral cavity (Beerens & Tahon-Castel, 1965;
Loubinoux *et al.*, 2002). However, their role in the
pathogenesis of periodontitis remains unclear. To
further investigate the potential pathogenic role of
*D. fairfieldensis* and *D. desulfuricans*, we studied
their potential ability to invade non-professional phag-
ocytic cells and their capacity to induce inflammatory
responses from these cells.

METHODS

Bacterial strains and tissue culture conditions

Strains used in this study were obtained from the
American Type Culture Collection (ATCC, Rockville,
MD): *D. desulfuricans* ATCC 29577, *D. desulfuricans*
ATCC 27774, *D. fairfieldensis* ATCC 700045, and
*P. gingivalis* ATCC 33277. All strains were stored at
-80°C in Brucella broth containing 15% glycerol
(weight/volume). Before testing, bacteria were grown
anaerobically at 37°C on Brucella agar supplemented
with 5% defibrinated sheep blood, for 24 h for *P. gin-
givalis* ATCC 33277 and 72 h for the *Desulfovibrio*
strains.

The KB cell line ATCC CCL-17 (HeLa cells) was
routinely cultured in flasks under a humidified atmo-
sphere containing 5% CO₂ at 37°C in RPMI-1640
medium (Gibco, Invitrogen, Cergy Pontoise, France)
supplemented with 10% fetal calf serum, 1% l-gluta-
mine (Gibco), 16 µg ml⁻¹ amikacin, and 128 µg ml⁻¹
amoxicillin.

Invasion assay

Invasion of epithelial cells was determined using an
antibiotic protection and invasion assay as previously
described by Han *et al.* (2000). For each strain, plate
cultures were harvested, washed twice in phosphate-
buffered saline (PBS) and suspended in unsupple-
mented cell culture medium at a concentration of
5 × 10⁷ cells ml⁻¹ determined by optical density. Bac-
terial suspensions (1 ml) were added to confluent KB
monolayers (multiplicity of infection 100 bacteria per
cell) in 24-well plates. After 2 h of incubation at 37°C,
unattached bacteria were removed by three washes
with unsupplemented RPMI-1640 medium. External
adherent cells were killed by incubating the infected
monolayers with fresh medium containing metronida-
zole (200 µg ml⁻¹) and gentamicin (300 µg ml⁻¹) for
1 h. The cell culture medium was removed and cells
were washed three times with PBS. Intracellular bac-
teria were then released by lysis of the cells in sterile
distilled water for 20 min. To assess intracellular per-
sistence and multiplication of *Desulfovibrio*, monolay-
ers infected with *D. fairfieldensis* ATCC 700045 and
*D. desulfuricans* ATCC 29577 were further incubated
in antibiotic-free and fetal calf serum-free medium for
24, 48, and 72 h. Before cell lysis, 0.1-ml aliquots of
the supernatant were sampled and examined for the
presence of extracellular bacteria. Serial dilutions of
the lysates were performed in sterile 0.9% NaCl, and
0.1-ml aliquots of the dilutions were plated onto
Brucella agar. Bacterial counts were expressed as
colony-forming units (CFU) per well.

Inhibitor assays of internalization

Inhibitor assays were performed using cytochalasin D (Sigma, Saint Quentin Fallavier, France), an actin microfilament polymerization inhibitor, and nocodazole (Sigma), a microtubule polymerization inhibitor. Stock solutions of inhibitors were prepared in dimethylsulfoxide and frozen at -20°C before assays. These solutions were diluted in unsupplemented RPMI-1640 to obtain final assay concentrations of 1 µg ml⁻¹ nocodazole and 10 µg ml⁻¹ cytochalasin D. The KB cells were incubated with cytochalasin D for 30 min before bacterial contamination. Nocodazole was added to the monolayers for 1 h on ice, and then warmed to 37°C for 30 min before the addition of bacteria. Each inhibitor was present during the assay.
The invasion assay was conducted as previously described. For each *Desulfoviribrio* strain tested, intra-cellular viable counts were determined at 2 h post-infection. At the concentrations used, inhibitors affected neither the viability of epithelial cells, as determined by examining the confluency of the monolayers and by trypan blue exclusion, nor the viability of bacterial strains as assessed by viable cell counting.

## Electron microscopy

For each *Desulfoviribrio* strain tested, ultrastructural studies of KB cells were performed at 2 h postinfection using transmission electron microscopy. Infected cell monolayers were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4). Following rinses with cacodylate buffer, the samples were postfixed for 1 h in 0.1 M cacodylate buffer containing 1% osmium tetroxide. The monolayers were dehydrated using increasing concentrations of ethanol [30, 50, 70, 80, and 90% (5 min for each step), 100% (20 min, three times)], and three changes of propylene oxide (20 min for each step). The samples were placed in a propylene oxide-epoxy resin (Epikote 812; Consortium International Pharmaceutique et Chimique, Paris, France) for 1 h at room temperature before being placed in 100% epoxy resin overnight as described by Luft *et al.* (1961). Then, they were embedded in fresh epoxy resin for 3 d at 56°C. Ultra-thin sections were cut on a Reichert OMU₃ ultramicrotome using a diamond knife, collected on copper grids, and stained with uranium acetate and Reynold's lead citrate. Sections were observed using a Philips CM12 M at 80 kV.

## Generation of antibodies

*Desulfoviribrio fairfieldensis* ATCC 700045 was cultured as previously described on supplemented Brucella blood agar for 72 h. Bacterial cells were washed three times with PBS, resuspended in sterile 0.9% NaCl at a final concentration of 10⁸ cells ml⁻¹ and finally inactivated using ultraviolet light. Inactivated bacteria were sent to Covalab Laboratory (Lyon, France) for the generation of polyclonal antibodies in rabbits. Specificity of the immune serum was evaluated by indirect immunofluorescence on *D. fairfieldensis* ATCC 700045, *D. desulfuricans*

ATCC 29577, *P. gingivalis* ATCC 33277, *F. nucleatum* Roger, *Prevotella intermedia* ATCC 25611, and *Prevotella buccae* ATCC 33574. Briefly, a 50-µl sample of cell suspension of each strain was spotted onto a slide for immunofluorescence tests, air dried, and fixed in cold ethanol. Smears were incubated with antiserum used at dilutions of 1 : 100, 1 : 200, or 1 : 500 for 30 min at 37°C in a moist chamber. After three washes in PBS, fluorescein isothiocyanate (FITC) -conjugated goat anti-rabbit immunoglobulin G antibody (Sigma) was added and incubated for another 30 min. After being washed with PBS, slides were examined with an Olympus fluorescence microscope at × 400 magnification. Specific reactivity was observed up to a dilution of 1 : 500 for both *Desulfoviribrio* strains. No cross-reactivity was found with any of the other strains tested.

## Confocal scanning laser microscopy

KB cells were seeded in eight-well Labteck II chambers (Nunc, Dutscher, France) at a concentration of 5 × 10⁴ cells well⁻¹ and incubated for 48 h. Before infection, KB cells were stained with 2 µm 1'-di-octadecyl 3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiIC₁₆) (Molecular Probe, Invitrogen, Cergy Pontoise, France) in RPMI-1640 for 2 min. DiIC₁₆ is a lipophilic carbocyanine membrane probe suitable for labeling cell surface membranes as well as intracytoplasmic membranes (Mukherjee *et al.*, 1999). Invasion assays were performed with *D. fairfieldensis* ATCC 700045 and *D. desulfuricans* ATCC 29577 as described above. At 2 h postinfection, cells were washed three times with unsupplemented RPMI-1640 medium and fixed in 2% paraformaldehyde at 4°C for 15 min. After three washes with PBS, cell monolayers were permeabilized with 0.1% Triton X-100 for 5 min. Cells were washed twice and incubated in PBS containing 5% bovine serum albumin (Sigma) for 20 min at room temperature (Kuehnel *et al.*, 2001). After removing the supernatant, cells were incubated overnight with rabbit anti-*Desulfoviribrio* serum diluted at 1 : 200. Cells were then incubated with an FITC-conjugated goat anti-rabbit immunoglobulin G (1 : 100) (Sigma) for 45 min followed by Alexa Fluor 488 anti-FITC-conjugated rabbit polyclonal immunoglobulin G (1 : 100) (Molecular Probe) for 45 min. Samples were observed with a confocal scanning laser
microscope (CLSM) SP2-AOBS (Leica Microsystems, Wetzlar, Germany), constituted by an inverted epifluorescence microscope equipped with an argon laser source at 488 nm and a helium/neon laser at 543 nm. The intensity of the fluorescence signal was measured between 500 and 520 nm for Alexa 488 (coded in green) and between 520 and 580 nm for Dil (coded in red) with a × 63 objective (water immersion/1.32 NA). For each frame, the collection of fluorescence signal was adjusted specifically to avoid the overlapping of the two emission dyes with a sequential mode. Different controls were achieved to eliminate non-specific detections. Uninfected cells with antibodies I, II, and III, and infected cells with antibodies II, III were used as negative controls to determine the threshold value of positive detection. Images were achieved with a z-step of 0.2 µm with a format of 512 × 512 pixels, 400 Hz, and analysed with Leica software Ics. Images were pseudocolored according to their respective emission and overloaded with Leica/Metamorph software.

## Cytokine induction assay

Culture media from 24, 48, and 72 h infected or uninfected KB cells were collected at the end of the experiments and stored at –20°C until assayed using enzyme-linked immunosorbent assay kits for IL-1β, IL-8 (RayBio, Norcross, GA), and IL-6 (Bender Medsystems, Vienna, Austria). Samples were processed according to the manufacturer's instructions. Plates were read at 450 nm using a microplate reader (Sunrise, Tecan, Lyon, France). The detection levels were 3, 8, and 0.98 pg ml⁻<fnref n="1" /> for IL-1β, IL-8, and IL-6, respectively. Cytokine concentrations were determined in triplicate by comparison with a standard curve prepared for each kit. Data were presented as means ± SD. Cytokine levels were compared using the unpaired Student's t-test (Graph Pad Software, San Diego, CA). Significances were defined as having *P* < 0.05.

# RESULTS

## Entry and survival of Desulfovibrio in KB cells

Two hours after infection, all three *Desulfovibrio* strains tested exhibited similar invasion efficiencies

<table><caption>**Table 1** Comparison of invasion of KB cells by *Desulfovibrio* spp. and *Porphyromonas gingivalis*</caption><thead><tr><th>Strain</th><th>CFU well<sup>-1</sup> recovered after antimicrobial treatment<sup>1</sup></th></tr></thead><tbody><tr><td><i>D. fairfieldensis</i> ATCC 700045</td><td>(1.53 ± 0.6) × 10<sup>4</sup></td></tr><tr><td><i>D. desulfuricans</i> ATCC 29577</td><td>(6.5 ± 2) × 10<sup>4</sup></td></tr><tr><td><i>D. desulfuricans</i> ATCC 27774</td><td>(3.81 ± 0.44) × 10<sup>4</sup></td></tr><tr><td><i>P. gingivalis</i> ATCC 33277</td><td>(3.86 ± 0.17) × 10<sup>5</sup></td></tr></tbody></table>

CFU, colony-forming units.

as assessed by the antibiotic protection assay (Table 1). However, the invasion efficiency of these strains (0.05–0.13% of the initial inoculum) was lower than those observed with *P. gingivalis* ATCC 33277 (0.69% of the initial inoculum), which was used as a positive control (Houalet-Jeanne *et al.*, 2001). For *D. fairfieldensis* ATCC 700045, the mean number of internalized viable bacteria was 3.5 times higher (*P* < 0.01) at 24 h postinfection than at 2 h postinfection, suggesting intracellular replication (Fig. 1). Thereafter, the viable intracellular counts gradually decreased (*P* < 0.01) down to about 57% of the initial intracellular count at 72 h postinfection. Similar results were found, as expected (Houalet-Jeanne *et al.*, 2001), for *P. gingivalis* ATCC 33277. For *D. desulfuricans* ATCC 29577, a statistically significant twofold increase (*P* < 0.01) of the mean number of viable intracellular bacteria occurred at 48 h. Thereafter, the intracellular counts decreased but remained slightly higher than those found at 2 h postinfection. The numbers provide evidence of an intracellular replication for *D. fairfieldensis* ATCC 700045 and a similar pattern is apparent for *D. desulfuricans* ATCC 29577 although the bacterial proliferation starts somewhat later. At no time, did the supernatant counts exceed 7% of the intracellularly recovered bacteria.

## Tubulin-dependent KB cells invasion by Desulfovibrio strains

Entry of bacteria into host cells requires microtubule or microfilament rearrangements (Meyer *et al.*, 1997). To assess whether internalization of *Desulfovibrio* strains is actin- and/or tubulin-dependent, we evaluated the effects of cytochalasin D and nocodazole

<footnote n="1">¹Values represent the means ± standard deviations from quadruplicate independent determination 2 h postinfection.</footnote>
<figure><img src="image_3.png" /><figcaption>**Figure 1** Intracellular viability of *Desulfoviribrio desulfuricans* ATCC 29577, *Desulfoviribrio fairfieldensis*, and *Porphyromonas gingivalis* ATCC 33277. Values represent the means ± SD from triplicate independent determinations at 2, 24, 48, and 72 h postinfection. Differences are statistically significant between connected values (\*P < 0.01).</figcaption></figure>

<table><caption>**Table 2** Effects of metabolic inhibitors on invasion of KB cells by *Desulfoviribrio desulfuricans* and *Desulfoviribrio fairfieldensis*</caption><thead><tr><th rowspan="2">Strains</th><th colspan="2">Percentage of invasion after treatment by<sup>1</sup></th></tr><tr><th>Nocodazole</th><th>Cytochalasin D</th></tr></thead><tbody><tr><td><i>D. fairfieldensis</i> ATCC 700045</td><td>23.3 ± 3.8</td><td>237.5 ± 39.2</td></tr><tr><td><i>D. desulfuricans</i> ATCC 29774</td><td>52.6 ± 1.9</td><td>138.3 ± 11.6</td></tr><tr><td><i>D. desulfuricans</i> ATCC 27774</td><td>10.7 ± 1.8</td><td>139.6 ± 11</td></tr></tbody></table>

Values represent the means ± standard deviations from triplicate independent determination 2 h postinfection.

¹The level of invasion was expressed as a percentage of the control level without any inhibitor.

pretreatment of KB cells on the internalization of the three *Desulfoviribrio* strains tested. Treatment of the KB cells with cytochalasin D resulted in increased numbers of internalized organisms at 2 h postinfection (Table 2). In contrast, for all three *Desulfoviribrio* strains tested, the counts of internalized viable bacteria at 2 h postinfection were lower in nocodazole-pretreated KB cells than in control cells, indicating that microtubule formation was required for cell invasion.

## Intracytoplasmic localization of *Desulfoviribrio* in KB cells

Once inside host cells, invasive bacteria replicate within endosomal organelles or escape from the endosomal pathway to the cytoplasm. At 2 h postinfection, TEM examination of KB cells infected with either *D. fairfieldensis* or *D. desulfuricans* showed that numerous bacteria adhered to the cell surface

<figure><img src="image_4.png" /><figcaption>**Figure 2** Transmission electron micrograph of *Desulfoviribrio desulfuricans* ATCC 29577 (arrow) inside the cytoplasm of a KB cell, at 2 h postinfection. Bar: 1 µm.</figcaption></figure>

while all observed intracellular bacteria appeared to be free in the cytoplasm without surrounding membranes (Fig. 2). The intracytoplasmic localization of both organisms tested was further confirmed by using confocal microscopy. Indeed, while numerous immunolabeled bacteria were shown to adhere to the epithelial cell surface 2 h postinfection, a few bacteria could be visualized within the cytoplasm of KB cells (Fig. 3). Internalized bacteria could be clearly differentiated from adherent bacteria by superimposing FITC-Alexia 488 and Dil images of serial sections (0.2 µm). No colocalization of intracellular immunolabeled bacteria with intracytoplasmic Dil-stained vesicles was observed suggesting that the observed intracellular bacteria were free in the cytoplasm.

## Production of cytokines by infected KB cells

The levels of IL-1β, IL-6, and IL-8 secreted by KB cells after infection with *D. desulfuricans* 29577 ATCC and *D. fairfieldensis* ATCC 700045 were also
<figure><img src="image_5.png" /><figcaption>**Figure 3** Invasion of KB cell by *Desulfovibrio fairfieldensis* ATCC 700045 visualized by CSLM. (A) *D. fairfieldensis* were stained green (arrow); (B) intracytoplasmic vesicles stained with Dil appeared red; (C) digital overlapping of images (A) and (B). No vesicles were colocalized with intracytoplasmic bacteria.</figcaption></figure>

measured. KB cells secreted significantly higher amounts of IL-8 at 2 h after infection with *D. fairfieldensis* ATCC 700045 (642 ± 65 pg ml⁻¹) or *D. desulfuricans* 29577 ATCC (744 ± 121 pg ml⁻¹) than uninfected KB cells (356 ± 73 pg ml⁻¹) (Fig. 4). This difference was no longer observed at 24, 48, and 72 h after infection with any of the strains tested. No IL-6 was detected in the supernatants of uninfected or infected cells at the 2-h time-point. KB cells constitutively expressed low levels of IL-6 at 24 h (2 ± 0.6 pg ml⁻¹), 48 h (2.4 ± 0.9 pg ml⁻¹), and 72 h (4.48 ± 1.9 pg ml⁻¹). In contrast, the amount of IL-6 released from KB cells infected with *D. desulfuricans* ATCC 29577 was significantly increased compared with the control cells at 24 h (4.1 ± 0.4 pg ml⁻¹) and 48 h (5.7 ± 0.7 pg ml⁻¹) (Fig. 5). When compared with uninfected cells, an increase, although not statistically significant, of IL-6 production was also observed for KB cells at 24 h (4.2 ± 1.5 pg ml⁻¹) and 48 h (4.7 ± 1.2 pg ml⁻¹) (*P* = 0.07) after infection

with *D. fairfieldensis* ATCC 700045. For both strains, IL-1β concentrations in supernatants from infected KB cells were found to be similar at each time-point studied to those measured in supernatants from control uninfected cells.

DISCUSSION

In the present study, we have shown that two putative periodontopathogens, *D. fairfieldensis* and *D. desulfuricans*, have the ability to invade oral epithelial cells. The intracellular location of *D. fairfieldensis* ATCC 700045 and *D. desulfuricans* ATCC 29577 was confirmed by both TEM and CSLM examination of infected monolayers. For these strains, 0.05–0.13% of the total number of bacteria initially added were recovered intracellularly at 2 h postinfection. For *P. gingivalis* ATCC 33277, 0.69% of the initial inoculum was recovered after lysis of the infected KB cells, which is very close to the 0.6% obtained by

<figure><img src="image_6.png" /><figcaption>**Figure 4** Secretion of interleukin-8 (IL-8) by KB cells at 2, 24, 48, and 72 h after infection with *Desulfovibrio desulfuricans* ATCC 29577 (Dd) and *Desulfovibrio fairfieldensis* ATCC 700045 (Df). The bars indicate the means and the standard deviations from three independent experiments. Statistically significant (*P* < 0.01) induction of cytokine production compared with control epithelial cells (C).</figcaption></figure>
<figure><img src="image_7.png" /><figcaption>**Figure 5** Secretion of interleukin-6 (IL-6) by KB cells at 2, 24, 48, and 72 h after infec- tion with *Desulfoviribrio desulfuricans* ATCC 29577 (Dd) and *Desulfoviribrio fairfieldensis* ATCC 700045 (Df). The bars indicate the means and the standard deviations from three independent experiments. Statistically significant (*P* < 0.01) induction of cytokine production compared with control epithelial cells (C).</figcaption></figure>

Houalet-Jeanne et al. (2001) who examined the inva-
sive potential of this isolate using similar experimen-
tal conditions. In our study, early internalization levels
observed with D. fairfieldensis and D. desulfuricans
were 5- to 15-fold lower than those observed with
P. gingivalis. However, to investigate the possibility
that invasion profiles are strain-specific more strains
need to be evaluated. Similar levels of internalization
were reported by Han et al. (2000) for another puta-
tive periodontopathogen, P. intermedia, the invasion
level of which has been reported as being 1/20 of
that of P. gingivalis 381 and F. nucleatum 12230.
Consequently, the Desulfoviribrio spp. strains tested
appeared to be weakly invasive. However, it cannot
be ruled out that other more invasive Desulfoviribrio
strains may exist or that interactions with other oral
bacteria may enhance the invasion potential of Des-
ulfoviribrio as shown for T. forsythia' interacting with
P. gingivalis (Inagaki et al., 2006). Also, it must be
stressed that KB cells, which are transformed cells,
are less efficient than epithelial cells from primary
gingival tissue cultures (Belton et al., 2004).

As previously reported by Houalet-Jeanne et al. (2001), we also observed that for *P. gingivalis* ATCC 33277, the number of intracellular viable bacteria increased about 1.5-fold at 24 h postinfection in infected KB cells. Quite similar results were also reported with another *P. gingivalis* strain (*P. gingivalis* FDC381) by Madianos et al. (1996), suggesting that *P. gingivalis* has the ability to multiply within KB cells. This could be of great clinical importance because Johnson et al. (2008) have demonstrated the presence of *P. gingivalis* within buccal epithelial cells. This suggests a mechanism by which bacteria might be protected from the host response and antimicro-

bial therapy, enabling them to re-colonize debrided
sites and perhaps contribute to the onset of refractory
disease. Likewise, host cells could constitute a reser-
voir of *Desulfoviribrio*. In the present study, the aver-
age number of intracellular viable bacteria increased
during the first 24 h for *D. fairfieldensis* ATCC
700045 and during the first 48 h for *D. desulfuricans*
ATCC 29577. These findings suggest that *Desulf-
ovibrio* is able to multiply within KB cells. Preliminary
experiments permitted us to ensure that the antibiotic
concentrations used were effective in killing the entire
bacterial inoculum within 1 h and that supernatant
bacterial counts remained low (≤7% of the intracellu-
lar counts) during the whole study period. Therefore,
the possibility of an invasion artefact is unlikely. How-
ever, the fact that some intracellular organisms may
exit the initially infected cells and then enter and mul-
tiply in new cells, as shown for other periodontopath-
ogens invading oral epithelial cells such as
*P. gingivalis* and *A. actinomycetemcomitans* (Meyer
et al., 1999; Yilmaz et al., 2006), cannot be ruled out
and needs further study.

In our study, TEM examination revealed that, at
2 h postinfection, all intracellular *Desulfoviribrio*
organisms appeared free within the cell cytoplasm.
Moreover, colocalization of intracellular bacteria
within intracytoplasmic vesicles was not found, sug-
gesting that *Desulfoviribrio* may escape the vacuole in
early stages of invasion as described for other patho-
gens (Meyer *et al.*, 1997; Houalet-Jeanne *et al.*,
2001).

For many bacterial pathogens, internalization by
epithelial cells is dependent on rearrangements of
the actin skeleton. Additionally, some pathogenic
bacteria enter epithelial cells via a microtubule-
dependent mechanism (Yoshida & Sasakawa,
2003). Among periodontopathogens, *P. gingivalis*
and *F. nucleatum* have been shown to enter epithe-
lial cells via both a microtubule-dependent and
actin-dependent mechanism (Lamont *et al.*, 1995;
Han *et al.*, 2000). It has also been reported that a
few strains of *A. actinomycetemcomitans* utilize
microtubule-dependent mode of invasion whereas
most strains use an actin-dependent mode of entry
into epithelial cells (Brissette & Fives-Taylor, 1999;
Meyer *et al.*, 1999). In the present study, treatment
with nocodazole, a microtubule-destabilizing agent,
resulted in a significant inhibition of bacterial inva-
sion whereas internalization was enhanced in cells
pretreated with cytochalasin D. These data suggest
that internalization of all three *Desulfovibrio* strains
tested was microtubule-dependent. It is noteworthy
that invasion of these strains was increased by cyto-
chalasin D. Similar observations were reported for
other organisms that require microtubule rearrange-
ment for invasion. This may be related to the fact
that cytochalasin D enhances lysosomal movement
to the cell periphery and hence invasion because of
the lysosome recruitment and fusion at the plasma
membrane (Tardieux *et al.*, 1992).

Besides bacterial internalization, inflammation
appears to be another important factor that may be
involved in the progression of periodontal infections
and local tissue destruction. It has been shown that
the production of various cytokines, including IL-6 and
IL-8, by oral epithelial cells following stimulation by pe-
riodontopathogens is an early event which participates
in the cascade of events leading to inflammation and
tissue destruction, and hence is critical for the progres-
sion of periodontitis (Graves, 2008). *In vitro* studies
have suggested that cytokine production by epithelial
cells may be triggered by their invasion by various bac-
terial pathogens including *P. gingivalis* (Sandros *et al.*,
2000; Eick *et al.*, 2006). Weglarz *et al.* (2007) demon-
strated that *D. desulfuricans* has the ability to induce
the secretion of IL-6 and IL-8 from the human colon
cell line Caco-2. In our study, both *Desulfovibrio*
strains tested stimulated the production of these
cytokines by KB cells. A significant increase in IL-8
secretion by epithelial cells was observed at 2 h post-
infection. This effect may be the result of various
mechanisms triggered by bacterial adhesion or inva-
sion and may play a role in the local immune response
as IL-8 acts as a chemokine directing neutrophil

migration to the infectious site. It cannot be excluded
that soluble products from *Desulfovibrio* strains directly
stimulated epithelial cells and induced upregulation of
IL-8, as described for *E. corrodens* (Yumoto *et al.*,
2007). In contrast, increased production of IL-6 was
only detected at 24 and 48 h after infection with *D. de-
sulfuricans* ATCC 29577 and, to a lesser extent, with
*D. fairfieldensis* ATCC 700045, suggesting that this
epithelial response might be more predominantly asso-
ciated with the presence of intracellular bacteria. IL-6
has also been identified as a periodontal disease mar-
ker and its release by host cells may participate in the
inflammatory reaction as well as in tissue destruction
via the activation of osteoclasts (Moreira *et al.*, 2007;
Herman *et al.*, 2008).

In conclusion, this study provides the first evi-
dence that *D. fairfieldensis* and *D. desulfuricans*, two
putative periodontopathogens, may invade oral epi-
thelial cells via a microtubule-dependent process.
We have also shown that, once inside the cells,
these bacteria had the capacity to remain viable for
prolonged periods and were able to elicit a minor
host cell response that may contribute to the devel-
opment of periodontal disease. Further studies of
these bacterium–host cell interactions are necessary
to increase our insight into the role of sulfate-reduc-
ing bacteria in periodontitis.

REFERENCES

Andrian, E., Grenier, D. and Rouabhia, M. (2004) *In vitro*
models of tissue penetration, destruction by *Porphyro-
monas gingivalis*. *Infect Immun* **72**: 4689–4698.

Beerens, H. and Tahon-Castel, M. (1965) *Infections Hu-
maines à Bactéries Anaérobies non Toxinogènes*. Brus-
sels: Presses Académiques Européennes.

Belton, C.M., Goodwin, P.C., Fatherazi, S., Schubert, M.M., Lamont, R.J. and Izutsu, K.T. (2004) Calcium oscillations in gingival epithelial cells infected with *Porphyromonas gingivalis*. *Microbes Infect* **6**: 440–447.

Boopathy, R., Robichaux, M., LaFont, D. and Howell, M. (2002) Activity of sulphate reducing bacteria in human periodontal pocket. *Can J Microbiol* **48**: 1099–1103.

Brissette, C.A. and Fives-Taylor, P.M. (1999) *Actinobacil-
lus actinomycetemcomitans* may utilize either actin-
dependent or actin-independent mechanisms of
invasion. *Oral Microbiol Immunol* **14**: 137–142.
Colombo, A.V., da Silva, C.M., Haffajee, A. and Colombo, A.P.V. (2007) Identification of intracellular oral species within human crevicular epithelial cells from subjects with chronic periodontitis by fluorescence in situ hybridization. *J Periodontal Res* **42**: 236–243.

Dorn, B.R., Leung, K.P. and Progulske-Fox, A. (1998) Invasion of human oral epithelial cells by *Prevotella intermedia*. *Infect Immun* **66**: 6054–6057.

Duncan, M.J., Nakao, S., Skobe, Z. and Xie, H. (1993) Interaction of *Porphyromonas gingivalis* with epithelial cells. *Infect Immun* **61**: 2260–2265.

Eick, S., Reissmann, A., Rödel, J., Schmidt, K.H. and Pfister, W. (2006) *Porphyromonas gingivalis* survives within KB cells and modulates inflammatory response. *Oral Microbiol Immunol* **21**: 231–237.

Gibson, G.R. (1990) Physiology and ecology of sulphate-reducing bacteria. *J Appl Bacteriol* **69**: 769–797.

Graves, D. (2008) Cytokines that promote periodontal tissue destruction. *J Periodontol* **79**: 1585–1591.

Han, Y.W., Shi, W., Huang, G.T.J. et al. (2000) Interactions between periodontal bacteria and human oral epithelial cells: *Fusobacterium nucleatum* adheres to and invades epithelial cells. *Infect Immun* **68**: 3140–3146.

Herman, S., Krönke, G. and Schett, G. (2008) Molecular mechanisms of inflammatory bone damage: emerging targets for therapy. *Trends Mol Med* **14**: 245–253.

Houalet-Jeanne, S., Pellen-Mussi, P., Tricot-Doleux, S., Apiou, J. and Bonnaure-Mallet, M. (2001) Assessment of internalization and viability of *Porphyromonas gingivalis* in KB epithelial cells by confocal microscopy. *Infect Immun* **69**: 7146–7151.

Inagaki, S., Onishi, S., Kuramitsu, H.K. and Sharma, A. (2006) *Porphyromonas gingivalis* vesicles enhance attachment, and the leucine-rich repeat BspA protein is required for invasion of epithelial cells by “Tannerella forsythia”. *Infect Immun* **74**: 5023–5028.

Johnson, J.D., Chen, R., Lenton, P.A., Zhang, G., Hinrichs, J.E. and Rudney, J.D. (2008) Persistence of extracrevicular bacterial reservoirs after treatment of aggressive periodontitis. *J Periodontol* **79**: 2305–2312.

Kuehnel, M.P., Goethe, R., Habermann, A. et al. (2001) Characterization of the intracellular survival of *Mycobacterium avium* ssp. paratuberculosis: phagosomal pH and fusogenicity in J774 macrophages compared with other mycobacteria. *Cell Microbiol* **3**: 551–566.

Lamont, R.J. and Yilmaz, O. (2002) In or out: the invasiveness of oral bacteria. *Periodontol 2000* **30**: 60–69.

Lamont, R.J., Chan, A., Belton, C.M., Izutsu, K.T., Vasel, D. and Weinberg, A. (1995) *Porphyromonas gingivalis*

invasion of gingival epithelial cells. *Infect Immun* **63**: 3878–3885.

Langendijk, P.S., Hagemann, J. and van der Hoeven, J.S. (1999) Sulfate-reducing bacteria in periodontal pockets and in health oral sites. *J Clin Periodontol* **26**: 596–599.

Langendijk, P.S., Hanssen, J.T. and Van der Hoeven, J.S. (2000) Sulfate-reducing bacteria in association with human periodontitis. *J Clin Periodontol* **27**: 943–950.

Langendijk, P.S., Kulik, E.M., Sandmeier, H., Meyer, J. and van der Hoeven, J.S. (2001) Isolation of *Desulfomicrobium orale* sp. nov. and *Desulfovibrio* strain NY682, oral sulfate-reducing bacteria involved in human periodontal disease. *Int J Syst Evol Microbiol* **51**: 1035–1044.

Langendijk-Genevaux, P.S., Grimm, W.D. and van der Hoeven, J.S. (2001) Sulfate-reducing bacteria in relation with other potential periodontal pathogens. *J Clin Periodontol* **28**: 1151–1157.

Loubinoux, J., Bisson-Boutelliez, C., Miller, N. and Le Faou, A.E. (2002) Isolation of the provisionally named *Desulfovibrio fairfieldensis* from human periodontal pockets. *Oral Microbiol Immunol* **17**: 321–323.

Luft, J.H. (1961) Improvements in epoxy resin embedding methods. *J Biophys Biochem Cytol* **9**: 409–411.

Madianos, P.N., Papapanou, P.N., Nannmark, U., Dahlen, G. and Sandros, J. (1996) *Porphyromonas gingivalis* FDC381 multiplies and persists within human oral epithelial cells *in vitro*. *Infect Immun* **64**: 660–664.

Meyer, D.H., Sreenivasan, P.K. and Fives-Taylor, P.M. (1991) Evidence for invasion of a human oral cell line by *Actinobacillus actinomycetemcomitans*. *Infect Immun* **59**: 2719–2726.

Meyer, D.H., Mintz, K.P. and Fives-Taylor, P.M. (1997) Models of invasion of enteric and periodontal pathogens into epithelial cells: a comparative analysis. *Crit Rev Oral Biol Med* **8**: 389–409.

Meyer, D.H., Rose, J.E., Lippmann, J.E. and Fives-Taylor, P.M. (1999) Microtubules are associated with intracellular movements and spread of the periodontopathogen *Actinobacillus actinomycetemcomitans*. *Infect Immun* **67**: 6518–6525.

Moreira, P.R., Lima, P.M., Sathler, K.O. et al. (2007) Interleukin-6 expression and gene polymorphism are associated with severity of periodontal disease in a sample of Brazilian individuals. *Clin Exp Immunol* **148**: 119–126.

Mukherjee, S., Soe, T.T. and Maxfield, F.R. (1999) Endocytic sorting of lipid analogues differing solely in the chemistry of their hydrophobic tails. *J Cell Biol* **144**: 1271–1284.

Rowan, F.E., Docherty, N.G., Coffey, J.C. and O’Connel, P.R. (2009) Sulphate-reducing bacteria and hydrogen
sulphide in the aetiology of ulcerative colitis. *Br J Surg* **2**: 151–158.

Sandros, J., Karlsson, C., Lappin, D.F., Madianos, P.N., Kinane, D.F. and Papapanou, P.N. (2000) Cytokine responses of oral epithelial cells to *Porphyromonas gingivalis* infection. *J Dent Res* **79**: 1808–1814.

Tardieux, I., Webster, P., Ravesloot, J. et al. (1992) Lyso-
some recruitment and fusion are early events required
for trypanosome invasion of mammalian cells. *Cell* **71**:
1117–1130.

Vianna, M.E., Holtgraewe, S., Seyfarth, I., Conrads, G. and Horz, H.P. (2008) Quantitative analysis of three hydrogenotrophic microbial groups, methanogenic archaea, sulfate-reducing bacteria, and acetogenic bacteria, within plaque biofilms associated with human peri-odontal disease. *J Bacteriol* **190**: 3779–3785.

Watanabe, K., Mikamo, H. and Tanaka, K. (2007) Clinical significance of sulfate-reducing bacteria for ulcerative colitis. *Nippon Rinsho* **65**: 1337–1346.

Weglarz, L., Wawszczyk, J., Orchel, A., Jaworska-Kik, M. and Dzierzewicz, Z. (2007) Phytic acid modulates *in vitro* IL-8 and IL-6 release from colonic epithelial cells stimulated with LPS and IL-1beta. *Dig Dis Sci* **52**: 93–102.

Willis, C.L., Gibson, G.R., Allison, C., Mac-Farlane, S. and Holt, J.S. (1995) Growth, incidence and activities of dissimilatory sulfate-reducing bacteria in the human oral cavity. *FEMS Microbiol Lett* **129**: 267–272.

Yilmaz, O., Verbeke, P., Lamont, R.J. and Ojcius, D.M. (2006) Intercellular spreading of *Porphyromonas gingivalis* infection in primary gingival epithelial cells. *Infect Immun* **74**: 703–710.

Yoshida, S. and Sasakawa, C. (2003) Exploiting host-
microtubule dynamics: a new aspect of bacterial inva-
sion. *Trends Microbiol* **11**: 139–143.

Yumoto, H., Yamada, M., Shinohara, C. et al. (2007) Soluble products from *Eikenella corrodens* induce cell proliferation and expression of interleukin-8 and adhesion molecules in endothelial cells via mitogen-activated protein kinase pathways. *Oral Microbiol Immunol* **22**: 36–45.

Zinkevich, V. and Beech, I.B. (2000) Screening of sul-
phate-reducing bacteria in colonoscopy samples from
healthy and colitic human gut mucosa. *FEMS Microbiol
Ecol* **34**: 147–155.
Copyright of Molecular Oral Microbiology is the property of Wiley-Blackwell and its content may not be
copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written
permission. However, users may print, download, or email articles for individual use.