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Localization of Porphyromonas gingivalis-carrying Fimbriae in situ in Human Periodontal Pockets
1 Department of Restorative Dentistry and Endodontology, Osaka University Graduate School of Dentistry, 1–8 Yamadaoka, Suita, Osaka 565-0871, Japan; and Correspondence: * corresponding author, noiri{at}dent.osaka-u.ac.jp
Fimbriae, which are involved in adherence, constitute an important pathogenic factor of Porphyromonas gingivalis. In vivo, however, the distribution of P. gingivalis-carrying fimbriae is unknown. The localization of P. gingivalis-carrying fimbriae was examined in situ. From 19 patients with severe periodontitis and P. gingivalis, we obtained 20 teeth with periodontal tissue attached, with and without immunolocalized fimbriae. Eleven teeth were subjected to light microscopy, 9 to electron microscopy. In 6 of the 11 samples examined, we detected positive reactions with an anti-P. gingivalis-fimbriae serum, located in the cementum-attached plaque area in the deep pocket zones. In the so-called ‘plaque-free zones’, P. gingivalis-carrying fimbriae were immunocytochemically observed to reside in contact with the dental cuticle in 6 of the 9 samples examined. These findings suggest that P. gingivalis-carrying fimbriae are strongly related to adherence to the root surface at the bottoms of human periodontal pockets.
Key Words: Porphyromonas gingivalis fimbriae • localization • human periodontal pockets • plaque-free zone • immunohistochemistry
Porphyromonas gingivalis, a major pathogen in severe forms of human periodontal diseases, possesses various virulence factors, including fimbriae (Lamont and Jenkinson, 1998). P. gingivalis fimbriae are specific cell-surface components involved in the adherence of this bacterium to various surfaces. Fimbriae were first purified from P. (previously Bacteroides) gingivalis strain 381, and their chemical, physical, morphological, and immunological properties have been characterized (Yoshimura et al., 1984, 1985). In their review, Lamont and Jenkinson (1998) described how P. gingivalis fimbriae can bind to other bacteria, saliva-coated hydroxyapatite, gingival epithelial cells, and extracellular matrix proteins in vitro. Their binding affinities for micro-organisms and substances are thought to foster the prevalence of P. gingivalis in human periodontal pockets. P. gingivalis appears to form small aggregates distributed throughout human periodontal pockets (Noiri et al., 1997) and is associated with plaque-biofilm formation at the bottoms of human periodontal pockets in the so-called ‘plaque-free zone’ (PFZ) (Noiri and Ebisu, 2000). However, the distribution of P. gingivalis-carrying fimbriae in situ has not yet been examined. The objective of the present study was to investigate the expression and localization of P. gingivalis fimbriae in human periodontal pockets.
Subjects and Site Selection Nineteen volunteers (16 males and three females, from 45 to 70 yrs of age) with advanced adult periodontitis participated in this study. Eleven specimens were prepared by a previously described method (Noiri et al., 1997, 2001) (Table 1  ). Nine teeth were extracted for examination under a transmission electron microscope (TEM) (Table 1). The criteria for all subjects and specimens have been described (Noiri et al., 1997). Informed consent was obtained from all patients according to the protocol approved by the Ethics Committee of the Osaka University Graduate School of Dentistry.
Strain and Culture Conditions The bacterial strains used included P. gingivalis 381, which was originally isolated from subgingival plaque by Dr. S.S. Socransky (Forsyth Institute, Boston, MA, USA) (Table 2 ). Mass cultures were grown anaerobically at 37°C as described previously (Yoshimura et al., 1984).
Preparation of Antisera against P. gingivalis Fimbriae and P. gingivalis A rabbit anti-P. gingivalis serum against total cell membrane from P. gingivalis strain 381 (Fimbriae genotype type I strain) was prepared and affinity-purified (Noiri et al., 1997). This antiserum can stain cell membranes. Its specificity with various oral bacteria has been shown previously (Noiri et al., 1997; Noiri and Ebisu, 2000). A rabbit antiserum against purified fimbriae from P. gingivalis 381 was prepared as described previously, with a pure preparation as the antigen (Yoshimura et al., 1984). Using immuno-TEM techniques, we examined the specificity of this anti-fimbriae serum against various P. gingivalis strains with different genotypes of fimbriae (Amano et al., 1999; Nakagawa et al., 2000) and other oral bacteria (Porphyromonas endodontalis ATCC 35406, Prevotella intermedia ATCC 25611, Fusobacterium nucleatum 1436, Streptococcus mutans MT8148, and Actinomyces naeslundii ATCC 12104) (Table 2  ). A fimbriae-deficient mutant from strain ATCC 33277, KDP98 (Watanabe-Kato et al., 1998), was kindly provided by Prof. Koji Nakayama (Nagasaki University Graduate School of Dentistry, Japan). Negative control samples obtained from periodontally healthy extracted teeth, and their surrounding periodontal tissues were prepared.
Immunohistochemical Procedure and Image Analysis Computer image analysis was carried out on the light microscopic images according to a previously described method (Noiri et al., 2001), and the immunolocalizations were then analyzed in human periodontal pocket specimens.
Preparation for Immuno-transmission Electron-microscopic Observation The sections were blocked with 20% normal goat serum in 0.1% bovine serum albumin (BSA) solution for 30 min, and then incubated with the anti-fimbriae serum diluted 1:100 in 0.1 M Tris-HCl-buffered saline (TBS) (pH 7.4) containing 1% BSA for 3 hrs at 4°C. After being washed, the samples were reacted with goat anti-rabbit IgG conjugated with 5 nm colloidal gold particles (GAR-G5; Amersham International plc, Buckinghamshire, UK), diluted 1:50 in 0.1% BSA-TBS. Subsequently, all the samples on the grids were fixed in 1% glutaraldehyde in a 0.1 M cacodylate buffer (pH 7.4), and stained with 1% aqueous uranyl acetate for 10 min and 1% lead acetate for 5 min. All samples were examined under a TEM (H-7500; Hitachi, Tokyo, Japan).
Specificity of Immunogold Labeling The anti-P. gingivalis serum reacted with all homologous bacterial cells examined (Table 2 ). The anti-fimbriae serum reacted with almost all P. gingivalis strains except for KDP98, a strain deficient in fimbriae, although strains W50 and W83 were previously reported to be afimbriate in biochemical tests (Suzuki et al., 1988). None of the other 5 bacterial species examined for cross-reactivity was labeled with the anti-fimbriae serum. In healthy periodontal tissues (negative controls), no background or any reaction with anti-P. gingivalis and anti-fimbriae was observed.
Localization of P. gingivalis-carrying Fimbriae in situ in Human Periodontal Pockets
P. gingivalis-carrying Fimbriae in the PFZ P. gingivalis 381 was treated with the anti-fimbriae serum, followed by immunogold staining (Fig. 2A ). The gold particles were distributed along the fimbriae. Positive reactions with the anti-fimbriae serum were detected in the PFZ in 6 of 9 samples examined. All positive reactions detected were observed on P. gingivalis attached to the dental cuticle, and fimbriae did not attach directly to the acellular cementum. Small numbers of bacteria, including different morphotypes, formed micro-colonies on the dental cuticle (DC) covering the acellular cementum (Fig. 2B). P. gingivalis carrying immunogold-labeled fimbriae was observed as small aggregates, as well as single cells, in close contact with the DC (Figs. 2C, 2D).
Using original techniques developed specifically for this study, we extended the investigation to detect P. gingivalis-carrying fimbriae, and found that P. gingivalis inhabiting human periodontal pockets appeared to express fimbriae. In vivo, there seems to be an association between the fimbrial content and some inflammatory cytokines in gingival crevicular fluid (Hirose et al., 1997). Since inflammatory cytokines are produced from the pocket epithelium, their concentrations in the epithelium-associated plaque area might be higher than in the other two areas. However, positive immunoreactions with the anti-fimbriae serum were mainly detected in the tooth-attached plaque area. The concentrations of inflammatory cytokines may not differ greatly among the cementum-attached, loosely attached, and epithelium-associated areas, since gingival crevicular fluid is constantly circulated in periodontal pockets. We do not yet know what local environmental factors are associated with the expression of fimbriae in vivo. Little attention has been paid to the DC on periodontitis-affected teeth, despite the fact that it possibly plays a key role in apical epithelial migration, progressive bacterial adhesion to the root surface, and protection of the exposed root (Friedman et al., 1993). We speculate that apical epithelial migration will occur by two pathways. The first is that bacteria inhabiting the bottoms of periodontal pockets and/or their toxins directly affect the junctional epithelium and periodontal ligament. The second is that subgingival bacteria attached to the pocket epithelium invade the gingiva, and then bacterial toxins and noxious products are transmitted via the gingival tissue and cause damage, eventually followed by deepening of the periodontal pockets. It is strongly suspected that P. gingivalis-carrying fimbriae, heavily located in the PFZ, could directly exhibit their pathogenicity on the junctional epithelium, because fewer samples were positive in the epithelium-associated plaque area, and no positive immunoreactions with the anti-fimbriae serum were observed within the pocket epithelial cells. The origin of the DC on periodontitis-affected cementum remains unclear. Kobayashi and Rose (1979) noted that the DC was a layer composed of highly condensed protein, since it was completely digested with the proteolytic enzymes trypsin and protease. Previous studies have suggested that the structure consists of anionic polymers, including glycoproteins (Friedman et al., 1993), or serum proteins, mainly albumin, IgA, and IgG, in the gingival fluid (Eide et al., 1984; López et al., 1990; Abbas et al., 1991). In contrast, purified P. gingivalis fimbriae bound strongly to glyco-conjugates, such as albumin-fucosylamide, through lectin-like interaction with carbohydrate (Sojar et al., 2004). P. gingivalis adherence to the DC might depend on P. gingivalis fimbriae and serum protein-carbohydrate interactions. The DC may also contain bacterial components (Eide et al., 1984). It has been suggested that bacterial adherence to the exposed root surface is mediated by the DC (Carrassi et al., 1989; Vrahopoulos et al., 1992; Friedman et al., 1993). A few studies have reported that bacterial adherence to cementum without prior subgingival pellicle formation could not be demonstrated (Carrassi et al., 1989; Friedman et al., 1993). P. gingivalis cells were detected in the PFZ (Mordan et al., 1999; Noiri and Ebisu 2000), and P. gingivalis-carrying fimbriae were also observed in the same zone in this study, indicating that fimbriae may play a crucial role in bacterial adherence to the DC. Results from the present study suggest that P. gingivalis fimbriae may possibly adhere to the DC rather than to other substances and organisms at the bottoms of human periodontal pockets. However, it remains unknown how the fimbriae mediate bacterial adherence to the DC. P. gingivalis has been considered to be a late colonizer and to bind to Gram-positive facultative bacteria that have already colonized as the first bacteria (Kolenbrander et al., 2002). However, at the bottoms of periodontal pockets, P. gingivalis appeared to play a role as an early colonizer in biofilm formation, although several types of obligate anaerobes were observed. In conclusion, we demonstrated that P. gingivalis fimbriae mediate adherence to the root surface at the bottoms of human periodontal pockets, rather than to the pocket epithelium.
This study was supported by Grants-in-Aid for Scientific Research (Nos. 14207080, 15591957, and 15592019) from the Japan Society for the Promotion of Science (JSPS), via the 21st century COE program of the JSPS, and the AGU High-Tech Research Center Project from the Ministry of Education, Culture, Sports, Science and Technology of Japan. Received for publication January 8, 2004. Revision received September 9, 2004. Accepted for publication September 21, 2004.
Journal of Dental Research, Vol. 83, No. 12,
941-945 (2004) This article has been cited by other articles:
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