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ISSN : 1226-7155(Print)
ISSN : 2287-6618(Online)
International Journal of Oral Biology Vol.46 No.3 pp.111-118
DOI : https://doi.org/10.11620/IJOB.2021.46.3.111

Inhibition of biofilm formation of periodontal pathogens by D-Arabinose

Sun-Jin An1, Jong-Uk Namkung1, Kyung-Won Ha2, Hye-Kyoung Jun2, Hyun Young Kim1, Bong-Kyu Choi1*
1Department of Oral Microbiology and Immunology, School of Dentistry, Seoul National University, Seoul 03080, Republic of Korea
2Bone Science R&D Center, OSSTEM IMPLANT Co., Ltd, Seoul 07789, Republic of Korea
*Correspondence to: Bong-Kyu Choi, E-mail: bongchoi@snu.ac.kr
August 26, 2021 September 1, 2021 September 1, 2021

Abstract


Periodontitis and periimplantitis are caused as a result of dental biofilm formation. This biofilm is composed of multiple species of pathogens. Therefore, controlling biofilm formation is critical for disease prevention. To inhibit biofilm formation, sugars can be used to interrupt lectin-involving interactions between bacteria or between bacteria and a host. In this study, we evaluated the effect of D-Arabinose on biofilm formation of putative periodontal pathogens as well as the quorum sensing activity and whole protein profiles of the pathogens. Crystal violet staining, confocal laser scanning microscopy, and scanning electron microscopy revealed that D-Arabinose inhibited biofilm formation of Porphyromonas gingivalis , Fusobacterium nucleatum , and Tannerella forsythia . D-Arabinose also significantly inhibited the activity of autoinducer 2 of F. nucleatum and the expression of representative bacterial virulence genes. Furthermore, D-Arabinose treatment altered the expression of some bacterial proteins. These results demonstrate that D-Arabinose can be used as an antibiofilm agent for the prevention of periodontal infections.


Periodontal pathogens , Biofilm inhibition , D-Arabinose , Quorum sensing

초록

    © The Korean Academy of Oral Biology. All rights reserved.

    This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/bync/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

    Introduction

    Periodontitis and periimplantitis are induced by biofilms in the subgingival crevice and periimplant crevice, respectively. Traditionally, putative pathogens of periodontitis and periimplantitis include Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, Filifactor alocis, Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, and Prevotella intermedia [1-4]. Periodontal pathogens are associated with diverse systemic diseases, including cardiovascular diseases, rheumatoid arthritis, colorectal cancer, pregnancy complications, osteoporosis, and Alzheimer’s disease [5,6]. Inhibition of biofilm formation of these pathogens is one of the most effective measures to prevent periodontal diseases and systemic diseases [7,8].

    Lectin-like protein adhesins and complementary polysaccharide- containing molecules on the cell surface contribute to bacterial coaggregation and the interaction of bacteria with the host [9]. Therefore, specific sugars can be used to interrupt these interactions via competitive binding [10]. Lactose, N-acetyl-Dgalactosamine, and D-galactose are known to inhibit coaggregation between many oral bacteria [11,12]. Additionally, some sugars act as quorum sensing inhibitors. Quorum sensing is a type of cell density-dependent communication between bacteria and induces biofilm formation and virulence [13,14]. DRibose and D-galactose have been shown to inhibit the biofilm formation of periodontal pathogens and the activity of intergenic quorum sensing molecule autoinducer 2 (AI-2) [15-17].

    In this study, we demonstrate that D-Arabinose inhibited biofilm formation of P. gingivalis, F. nucleatum, and T. forsythia as well as the activity of F. nucleatum AI-2. We also showed that treatment with D-Arabinose altered the expression of representative surface proteins in the bacteria.

    Materials and Methods

    1. Bacterial culture

    P. gingivalis (ATCC 33277) and F. nucleatum (ATCC 25586) were cultured anaerobically (10% H2, 10% CO2, 80% N2) in brain heart infusion broth supplemented with hemin (10 μg/ mL) and vitamin K (0.2 μg/mL) at 37℃. T. forsythia (ATCC 43037) was grown in New Oral Spirochete broth (ATCC medium 1494) supplemented with N-acetylmuramic acid (0.01 μg/ mL) and vitamin K (0.02 μg/mL) under anaerobic conditions. The AI-2 reporter strain Vibrio harveyi BB170 was aerobically cultured in autoinducer bioassay (AB) medium consisting of 0.3 M sodium chloride, 0.05 M magnesium sulfate, 0.2% casamino acids, 10 mM potassium phosphate (pH 7.0), 1 mM Larginine, and 2% glycerol at 30℃ with shaking.

    2. Effect of D-Arabinose on growth of planktonic bacteria

    P. gingivalis, F. nucleatum, and T. forsythia were grown anaerobically in the presence or absence of D-Arabinose (50 and 100 mM). Bacterial growth was monitored for up to 48–60 hours by measuring the absorbance at 600 nm using a microplate reader (EPOCH2 Absorbance Microplate Reader; BioTek, Winooski, VT, USA).

    3. Biofilm formation assay

    Biofilm formation was evaluated by crystal violet staining. Each bacterial suspension was added to 24-well plates with a cover slip (12 mm × 12 mm) in the absence or presence of DArabinose and incubated for a total of 48 hours under anaerobic conditions (10% H2, 10% CO2, 80% N2). The numbers of P. gingivalis, F. nucleatum, and T. forsythia added were 5 × 108, 3 × 108 and 1 × 109 bacteria/mL, respectively. After 24–48 hours, biofilms that formed on the cover slips were washed with phosphate buffered saline (PBS) and stained with 1% crystal violet [Tris(4-imethylamino)phenyl)methylium chloride] for 15 minutes. After washing twice with PBS, crystal violetstained biofilms on the cover slips were destained with 1 mL of acetone-alcohol (20:80, vol/vol). The absorbance at 590 nm of the solution containing crystal violet was measured by a microplate reader (EPOCH2 Absorbance Microplate Reader).

    The effect of D-Arabinose pretreatment on biofilm formation was also evaluated. Cover slips were placed into a solution containing 50 mM and 100 mM D-Arabinose for 3 minutes. After removing the solution, bacteria were added onto cover slips and incubated for 24–48 hours. The biofilms formed were stained with 1% crystal violet and measured as described above.

    The biofilms formed on the cover slips were stained using the Live/Dead-BacLight bacterial viability kit (Invitrogen, Carlsbad, CA, USA) and observed using a confocal laser scanning microscope (CLSM, LSM 700; Carl Zeiss, Jena, Germany) at a magnification of 630×. Additionally, biofilms were prefixed with PBS containing 2.5% glutaraldehyde and 2% paraformaldehyde overnight. After washing three times with PBS, bacteria on the cover slips were fixed with 1% osmium tetroxide for 2 hours. The cover slips were washed three times with PBS, dehydrated in an ascending ethanol series (70%, 80%, 90%, 95%, and 100% each for 15 minutes) and coated with platinum using a sputter-coater (Turbomolecular pump coating system, Q150T S; Quorum Technologies, Laughton, East Sussex, UK) at a sputter current of 20 mA for 360 seconds in vacuum and observed by field emission scanning electron microscopy (SEM, Apreo S; Thermo Fisher Scientific, Waltham, MA, USA) at a magnification of 30,000×.

    4. Effect of D-Arabinose on F. nucleatum AI-2 activity

    The effect of D-Arabinose on AI-2 activity was determined using partially purified F. nucleatum AI-2 and the AI-2 reporter strain V. harveyi BB170, which expresses luciferases, as previously described [16]. V. harveyi BB170 (1 × 107 bacteria/mL) in AB medium was mixed with 10% (vol/vol) F. nucleatum AI-2 in the presence or absence of D-Arabinose and incubated for 6 hours at 30℃ under aerobic conditions. The bioluminescence was measured with a luminometer (Synergy H1-Multi Mode Microplate Reader; BioTek).

    5. Quantitative polymerase chain reaction (qPCR)

    The effect of D-Arabinose on the expression of representative adhesins of P. gingivalis , F. nucleatum , and T. forsythia was analyzed by qPCR as described previously [16]. The se quences of the primers used were as follows: 5’-ACG CTT CCC ATT CTA TCA CG-3’ and 5’- GAG GGT GCA ATC AGG ACA TT-3’ for rgpA; 5’- ACT CGT ATC GCC CGT TAT TC-3’ and 5’- TGC AAC TTG CCG TAC AGA GGG-3’ for P. gingivalis 16S rRNA; 5’-TGT TTC TGC TTC AGC ATT CG-3’ and 5’-GTG CTT GTC TAG CAG CGT CA-3’ for fadA; 5’-TGG AGG TCC GCT TAC CTC TCC AG-3’ and 5’-AAG CGC GTC TAG GTG GTT ATG T-3’ for F. nucleatum 16S rRNA; 5’-GAG AGC GCC TTT TAC GAT TG-3’ and 5’-GTG GTT ACG CTG TTC GGA AT-3’ for bspA; and 5’-TTA CCT GTT AGC AAC TGA CAG TCG-3’ and 5’-ATT GAA ATG TAG ACG ACG GAG AGT-3’ for T. forsythia 16S rRNA. The 16S rRNA gene was used as a reference for normalization of the gene expression levels.

    6. Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of D-Arabinosetreated bacteria

    Bacterial cultures (20 mL each) in the presence or absence of D-Arabinose were harvested by centrifugation at 10,000 × g for 10 minutes at 4℃. The bacterial pellets were lysed using RIPA buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, 50 mM NaF, 1 mM EDTA, 5 mM Na3VO4) and 1 mM phenylmethylsulfonyl fluoride and 1 × protease inhibitor cocktail (Roche, Basel, Switzerland), and 100 μg/mL lysozyme. The lysates were centrifugation at 13,000 × g for 45 minutes at 4℃. Protein concentrations of the lysates were determined using the bicinchoninic acid assay (Thermo Fisher Scientific). The supernatants (10 µg protein) were subjected to SDS-PAGE (12% polyacrylamide gel) and stained with a SYPRO® Ruby Protein Gel Stain kit (Thermo Fisher Scientific). The protein gel was detected using the Gel-doc system (VILBER; Scintica, Webster, TX, USA).

    7. Statistical analysis

    Statistical analyses were performed using the Mann–Whitney U test or the Kruskal–Wallis test. Statistically significant differences between the control and sugar-treated groups were analyzed. A p-value less than 0.05 was considered to be statistically significant.

    Results

    1. Inhibitory effect of D-Arabinose on biofilm formation of periodontal pathogens

    We first tested whether D-Arabinose affects planktonic bacterial growth. D-Arabinose at 50 and 100 mM did not affect the growth of P. gingivalis and F. nucleatum , while DArabinose at 100 mM reduced T. forsythia growth (Fig. 1A). D-Arabinose was evaluated for its inhibitory effect on the biofilm formation of periodontal pathogens on cover slips. The biofilm formation of P. gingivalis, F. nucleatum, and T. forsythia was significantly inhibited in the presence of 50 and 100 mM D-Arabinose (Fig. 1B). Pretreatment of cover slips with DArabinose for 3 minutes also significantly reduced bacterial biofilm formation, except for P. gingivalis at 50 mM arabinose (Fig. 1B). The inhibitory effect of D-Arabinose on the biofilm formation of P. gingivalis, F. nucleatum, and T. forsythia was visualized by CLSM after live/dead staining (Fig. 2A) and SEM (Fig. 2B).

    2. Inhibition of F. nucleatum AI-2 activity by D-Arabinose

    F. nucleatum AI-2 plays an important role in the development of subgingival biofilms. Previously, we reported that Dgalactose inhibited AI-2 activity in F. nucleatum [17]. In this study, we assessed whether D-Arabinose inhibited F. nucleatum AI-2 using the AI-2 reporter strain V. harveyi BB170 by measuring bioluminescence. D-Arabinose at 50 and 100 mM significantly inhibited F. nucleatum AI-2 activity at 3 and 6 hours incubation time points (Fig. 3).

    3. Inhibition of expression of virulence factors by D-Arabinose

    AI-2 has been known to increase the expression of bacterial virulence factors, and AI-2 inhibitors are expected to reduce the expression of virulence factors of periodontal pathogens. Therefore, we tested whether D-Arabinose inhibited the expression of virulence factors that are known to be adhesins and immunomodulators. When the bacteria were treated with 50 mM D-Arabinose, the gene expression of the gingipain RgpA of P. gingivalis and FadA of F. nucleatum was significantly decreased, as analyzed by qPCR (Fig. 4). The gene expression of T. forsythia BspA was not significantly decreased by DArabinose treatment but showed a tendency to decrease.

    4. Modification of protein profiles of periodontal pathogens by D-Arabinose

    As D-Arabinose, an AI-2 inhibitor, reduced representative virulence factors of P. gingivalis, F. nucleatum, and T. forsythia, we compared the whole protein profiles of the bacteria in the presence or absence of D-Arabinose by SDS-PAGE. As shown in Fig. 5, D-Arabinose treatment modified the protein profiles of P. gingivalis, F. nucleatum, and T. forsythia.

    Discussion

    This study demonstrated that D-Arabinose inhibited the biofilm formation of periodontal pathogens and AI-2 activity. Additionally, D-Arabinose reduced the gene expression of representative surface virulence factors of the periodontal pathogens and modified the protein profiles of the bacteria. Inhibition of biofilm formation is critical for the prevention of periodontitis and periimplantitis. Lectin-like proteins are involved in adhesion to bacteria and hosts by binding carbohydrate residues in partner molecules. Some sugars can compete with lectin binding interactions. Lactose, galactose, fucose, ribose, and galactosamine have been shown to inhibit coaggregation between bacteria [10,16,17]. In addition, galactose and ribose have been suggested to compete with AI-2 receptors, resulting in reduced AI-2 activity of F. nucleatum and A. actinomycetemcomitans [15-17]. D-Ribose inhibited the gene expression of P. gingivalis gingipain RgpA, F. nucleatum FadA, T. forsythia BspA, and T. denticola Msp, which were induced by AI-2 purified from F. nucleatum [16]. In the present study, we showed that D-Arabinose inhibited P. gingivalis gingipain RgpA, F. nucleatum FadA, and T. forsythia BspA. F. nucleatum FadA is involved in attachment to host epithelial cells and induces inflammation [18]. P. gingivalis gingipain RgpA is an arginine-specific cysteine proteinase with adhesion ability to oral epithelial cells and other bacterial species [19]. T. forsythia BspA is a glycosylated protein associated with the outer membrane with a leucine-rich-repeat motif [20]. The BspA protein adheres to epithelial cells and host proteins, including fibronectin and fibrinogen. It is also involved in coaggregation with other oral bacteria and induces inflammation [21]. SDS-PAGE analyses revealed that D-Arabinose modulated the expression of diverse proteins. Therefore, it may be interesting to identify proteins demonstrating differential expression in the presence of D-Arabinose by proteomic analysis.

    In addition to the periodontal pathogens included in this study, D-Arabinose was shown to inhibit the biofilm formation of oral bacteria in the saliva of healthy subjects while not affecting the planktonic growth of oral streptococci, including Streptococcus gordonii , Streptococcus mitis , Streptococcus oralis, and Streptococcus sanguinis [22].

    In summary, D-Arabinose showed an inhibitory effect on biofilm formation of P. gingivalis, F. nucleatum, and T. forsythia. It inhibited F. nucleatum AI-2 activity and the expression of representative adhesins of the bacteria. Three minutes of pretreatment also showed significant inhibition of biofilm formation. These results indicate that D-Arabinose can be used as an antibiofilm agent for the prevention of periodontal infections.

    Acknowledgements

    This work was supported by the World Class 300 Project (R&D) from Korea Institute for Advancement of Technology (grant number P108100012) and the Dental Research Institute of Seoul National University.

    Figure

    IJOB-46-3-111_F1.gif

    Effects of D-Arabinose (D-Ara) on bacterial growth. (A) Porphyromonas gingivalis , Fusobacterium nucleatum , and Tannerella forsythia were grown under anaerobic conditions at 37℃ in the presence of 50 and 100 mM D-Arabinose. Bacterial growth was monitored for 48–60 hours by measuring the optical density (OD) at 600 nm using a spectrophotometer. (B) P. gingivalis (5 × 108 bacteria/mL), F. nucleatum (3 × 108 bacteria/mL), and T. forsythia (1 × 109 bacteria/mL) were grown in the presence of D-Arabinose on cover slips under anaerobic conditions for 24–48 hours at 37℃. The biofilms formed were stained with 1% crystal violet for 15 minutes and destained with 1 mL of acetone-alcohol (20:80, vol/vol). The OD of the destaining solution containing crystal violet was measured at 590 nm. Separately, cover slips were pretreated with D-Arabinose for 3 minutes, and then P. gingivalis (5 × 108 bacteria/mL), F. nucleatum (3 × 108 bacteria/mL), or T. forsythia (1 × 109 bacteria/mL) was added to the coverslips. After incubation for 24–48 hours, the biofilms formed on the cover slips were analyzed as described above.

    *p < 0.05 compared to the nontreated control.

    IJOB-46-3-111_F2.gif

    Biofilm images of confocal laser scanning microscope and scanning electron microscopy (SEM). (A) The biofilms of Porphyromonas gingivalis (Pg), Fusobacterium nucleatum (Fn), and Tannerella forsythia (Tf) that formed on the cover slips were stained with the Live/Dead-BacLight bacterial viability kit and observed under a confocal laser scanning microscope at a magnification of 630×. The scale bar indicates a length of 10 μm. (B) The biofilms formed on the cover slips were observed by SEM at a magnification of 30,000×. The scale bar indicates a length of 3 μm. D-Ara 50 mM: D-Arabinose was present over the whole incubation period, or cover slips were pretreated with D-Arabinose for 3 minutes before adding bacteria.

    IJOB-46-3-111_F3.gif

    Inhibition of Fusobacterium nucleatum (Fn) autoinducer 2 (AI-2) activity by D-Arabinose (D-Ara). Vibrio harveyi BB170 (1 × 107 bacteria/mL) was incubated with 10% (vol/vol) AI-2 of F. nucleatum in the presence or absence of D-Arabinose. The bioluminescence at the 3-hour and 6-hour time points was measured using a luminometer. The data are the mean ± standard deviation of a representative experiment.

    *p < 0.05 compared to the nontreated control, #p < 0.05 compared to the F. nucleatum AI-2-treated control.

    IJOB-46-3-111_F4.gif

    Effect of D-Arabinose (D-Ara) on gene expression of adhesion molecules of bacteria. Porphyromonas gingivalis , Fusobacterium nucleatum , and Tannerella forsythia were cultured in the presence or absence of D-Arabinose. Bacterial RNA was isolated, and the mRNA expression of bacterial adhesion molecules was analyzed by quantitative polymerase chain reaction. Gene expression is presented as the relative ratio compared with that of 16S rDNA.

    *p < 0.05 compared to the nontreated control.

    IJOB-46-3-111_F5.gif

    Protein profiles following treatment with D-Arabinose (D-Ara). Porphyromonas gingivalis , Fusobacterium nucleatum , and Tannerella forsythia were cultured in the presence or absence of D-Arabinose. The protein profiles were analyzed by sodium dodecylsulfate polyacrylamide gel electrophoresis (each 10 µg) and luminescent staining using a Ruby Protein Gel Stain kit (Thermo Fisher Scientific, Waltham, MA, USA). Closed arrow heads indicate proteins that were decreased by D-Arabinose treatment and open arrow heads indicate proteins that were increased by D-Arabinose.

    Table

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