In Situ Intraepithelial Localizations of Opportunistic Pathogens, Porphyromonas gingivalis and Filifactor alocis, in Human Gingiva

Abstract

Porphyromonas gingivalis and Filifactor alocis are fastidious oral pathogens and etiological agents associated with chronic periodontitis. Although previous studies showed increased levels of the two obligate anaerobic species in periodontitis patients, methodologies for this knowledge were primarily limited to sampling subgingival plaque, saliva, or gingival crevicular fluid. To evaluate the extent to which P. gingivalis and F. alocis may invade the periodontal tissues, an in situ cross-sectional study was comparatively conducted on the gingival biopsy specimens of patients diagnosed with periodontal health or chronic periodontitis. Immunostained tissue sections for each organism were imaged by Super-Resolution Confocal Scanning Microscopy to determine the relative presence and localization of target bacterial species. Fluorescence-in-situ-hybridization (FISH) coupled with species specific 16S rRNA method was utilized to confirm whether detected bacteria were live within the tissue. In periodontitis, P. gingivalis and F. alocis revealed similarly concentrated localization near the basement membrane or external basal lamina of the gingival epithelium. The presence of both bacteria was significantly increased in periodontitis vs. healthy tissue. However, P. gingivalis was still detected to an extent in health tissue, while only minimal levels of F. alocis were spotted in health. Additionally, the micrographic analyses displayed heightened formation of epithelial microvasculature containing significantly co-localized and metabolically active dual species within periodontitis tissue. Thus, this study demonstrates, for the first-time, spatial arrangements of P. gingivalis and F. alocis in both single and co-localized forms within the complex fabric of human gingiva during health and disease. It also exhibits critical visualizations of co-invaded microvascularized epithelial layer of the tissue by metabolically active P. gingivalis and F. alocis from patients with severe periodontitis. These findings collectively uncover novel visual evidence of a potential starting point for systemic spread of opportunistic bacteria during their chronic colonization in gingival epithelium.

Keywords: Dual species colonization in gingiva; Filifactor alocis; Metabolically active bacteria in human tissue; Periodontal epithelium; Porphyromonas gingivalis.

Graphical abstract

Fig. 1

Detection of P. gingivalis in gingival tissues in health vs. periodontitis. (A) Representative images of H&E stained gingival biopsy specimens from healthy individuals and periodontitis patients. 10x magnification. (B) The specificity of anti-P. gingivalis antibody is shown in the fluorescence micrograph presenting P. gingivalis (green) in human primary gingival epithelial cells (GECs) infected with the microorganism for 24 h. 40x magnification. (C) Mean fluorescence intensity (MFI) of P. gingivalis detection from gingival tissue sections in health and periodontitis obtained by confocal microscopy. Data are presented as mean ± SD. Representative images from at least 5 different patients per group were used for quantitative analysis. (D) Representative confocal images of gingival biopsy specimens from healthy individuals (i-v) and periodontitis patients (vi-x). (i, vi) 1:500 DAPI staining to visualize cellular DNA. (ii, vii) P. gingivalis detection using 1:200 rabbit anti-P. gingivalis primary antibody and 1:500 Alexafluor-488 (Green) conjugated goat anti-rabbit secondary antibody. 63x magnification with oil immersion. (iii-v, viii-x) DAPI (Blue), P. gingivalis, and CD73 of the gingival epithelium for a counterstaining were merged. A mouse antibody against CD73 coupled with Alexafluor-568 (Red) conjugated goat anti-mouse secondary antibody was used to outline the borders of the tissues. Images were captured using super resolution confocal laser scanning microscopy at 63x magnification with oil immersion. The range of z-stacks was kept consistent. (E) Representative three-dimensional views of z-stacks of the gingival tissue in periodontitis shown in (D; viii) to show the spatial information of the immunostained tissues. SC: Stratum corneum, SL: Stratum lucidum, SG: Stratum granulosum, SB: Stratum basale, LT: Lamina propria. Scale bars in A, B, and D = 20µm.

Fig. 2

Detection of F. alocis in gingival tissues in health vs. periodontitis. (A) The specificity of anti-F. alocis antibody is shown in the confocal micrograph presenting F. alocis (green) in human primary gingival epithelial cells (GECs) infected with the microorganism for 24 h. (B) Mean fluorescence intensity of F. alocis detection from gingival tissue sections in health and periodontitis obtained by confocal microscopy. Data are presented as mean ± SD. Representative images from at least 5 different patients per group were used for quantitative analysis. (C) Representative confocal images from health individuals displaying upper gingival epithelium (i-iii) and lower gingival epithelium tissue sections (iv-vi). (D) Representative confocal images from periodontitis patients displaying upper gingival epithelium (i-v) and lower gingival epithelium tissue sections (vi-x). 1:500 DAPI staining to visualize cellular DNA (C; i, iv and D; i, vi). F. alocis detection using 1:200 rabbit anti-F. alocis primary antibody and 1:500 Alexafluor-488 (Green) conjugated goat anti-rabbit secondary antibody. 63x magnification with oil emersion (C; ii, v, D; ii, vii). DAPI (Blue), F. alocis (Green), and CD73 of the gingival epithelium for a counterstaining were merged (C; iii, vi, D; iii-v, viii-x). A mouse antibody against CD73 coupled with Alexafluor-568 (Red) conjugated goat anti-mouse secondary antibody was used to outline the borders of the tissues. Images were captured using super resolution confocal laser scanning microscopy at 63x magnification with oil immersion. The range of z-stacks was kept consistent. (E) Representative three-dimensional views of z-stacks of the gingival tissue in periodontitis shown in (D, iii) to show the spatial information of the immunostained tissues.SC: Stratum corneum, SL: Stratum lucidum, SG: Stratum granulosum, SB: Stratum basale, LT: Lamina propria. Scale bars in A, C, and D = 20µm.

Fig. 3

Double staining of co-localized P. gingivalis and F. alocis in gingival tissue from periodontitis patients and detection of vascular structures in the epithelial layer of the periodontitis tissue. (A) Representative images of immunostained tissues from healthy individuals and periodontitis patients are shown. (i) The entrance to the sulcus on the left and connective tissue attachment on the right is with a white arrow. 1:500 DAPI staining to visualize cellular DNA. (ii) P. gingivalis detection using 1:200 mouse anti-P. gingivalis primary antibody and 1:500 Alexafluor-488 (Green) conjugated goat anti-mouse secondary antibody. (iii) F. alocis detection by using 1:200 rabbit anti-F. alocis primary antibody and 1:500 Alexafluor-568 (Red) conjugated goat anti-rabbit secondary antibody. (iv) DAPI (Blue), P. gingivalis, and F. alocis merged for co-localization with an average Pearson correlation coefficient of 0.88 via the ImageJ with JaCoP plug-in. A representative blood vessel-like structure detected is noted with a white arrow. (v-viii) Zoomed-in images highlighting a blood vessel like structure. 20x magnification. (B) The presence of microvasculature in the gingival epithelium of participants of healthy and periodontitis group in percentages. At least 3 separate sectioned slides were examined for each subject's tissue (presence or absence) and the frequency of gingival microvasculature was averaged within the group. Data are presented as mean ± SD.(C) Representative H&E stained images of gingival tissues from periodontitis patients showing heightened formation of microvasculature in the gingival epithelium. 5x (i) and 10x (ii) magnification. Black arrows indicate representative gingival microvasculature. Representative confocal microscopy images of immunostained tissue sections from gingival biopsies taken from patients diagnosed with severe chronic periodontitis. (iii) H&E staining to show tissue structure. (iv) P. gingivalis detection using 1:200 rabbit anti-P. gingivalis primary antibody and 1:500 Alexafluor-488 (Green) conjugated goat anti-rabbit secondary antibody. (v) A vascular marker CD31 antibody (mouse, monoclonal) staining followed by Alexafluor-568 (Red) conjugated goat anti-mouse secondary antibody. Arrows point to intense CD31 red staining around microvessels (vi) P. gingivalis (Green) and CD31 (Red) merged (vii) A zoomed-in image highlight a microvasculature in the gingival epithelium and the arrows point to high presence of P. gingivalis within the blood vessel. At 20x (A) and 63x (C) magnification with oil immersion. SC: Stratum corneum, SL: Stratum lucidum, SG: Stratum granulosum, SB: Stratum basale, LT: Lamina propria. Scale bars in both A and C = 20µm.

Fig. 4

Co-localization of P. gingivalis and F. alocis within the gingival microvasculature. (A) Representative confocal images of the gingival epithelium (i-v) and lamina propria (vi-x) from periodontitis patients are shown. (i, vi) 1:500 DAPI staining to visualize cellular DNA. (ii, vii) P. gingivalis detection using 1:200 mouse anti-P. gingivalis primary antibody and 1:500 Alexa Fluor 488 (Green) conjugated goat anti-mouse secondary antibody. (iii, viii) F. alocis detection by using 1:200 rabbit anti-F. alocis primary antibody and 1:500 Alexa Fluor 568 (Red) conjugated goat anti-rabbit secondary antibody. (iv, ix) DAPI (Blue), P. gingivalis (Green), and F. alocis (Red) merged. The white arrow indicates P. gingivalis and F. alocis co-localization. (v, x) Zoomed-in images highlighting co-aggregated P. gingivalis and F. alocis (yellow) invading vasculature in the gingival epithelium with an average Pearson correlation coefficient of 0.92 via the ImageJ with JaCoP plug-in. 63x magnification with oil immersion. SC: Stratum corneum, SL: Stratum lucidum, SG: Stratum granulosum, SB: Stratum basale, LT: Lamina propria. Scale bars = 20µm. (B) The number of microvessels with and without both bacteria out of all microvessels present within the same tissue in percentages. Each periodontitis tissue presenting robust gingival microvasculature was examined. The number of microvessels with the bacteria, without the bacteria, and total number of vessels present were recorded for quantification. Data are presented as mean ± SD.

Fig. 5

Co-colonization of metabolically active P. gingivalis and F. alocis within the gingival tissue in periodontitis. (A) Representative micrographs of live P. gingivalis and F. alocis within the epithelial layer (i-ix) and lamina propria (x-xiii) of gingiva sections from patients with chronic periodontitis via FISH method as previously described (Velsko et al., 2014, Schlafer et al., 2010). Hybridization was performed using P. gingivalis 16S rRNA-specific oligonucleotide POGI labeled with Alexafluor 488 (Green fluorescent) and F. alocis 16S rRNA-specific oligonucleotide FIAL labeled with Alexafluor-594 (Red fluorescent) probes. (i, vi, x) 1:500 DAPI (Blue) staining to visualize cellular DNA. (ii, vii, xi) Metabolically active P. gingivalis detection using 5nM P. gingivalis specific probe tagged with Alexafluor-488. (iii, viii, xiii) Metabolically active F. alocis detection using 5nM F.a specific probe tagged with Alexafluor-594. (iv, ix, xiii) DAPI, P. gingivalis, F. alocis merged. The white arrows indicate both bacterial species are invading deeper into the tissue in mixed colonies through the epithelial tissue. Co-localization of the two species is shown in yellow with an average Pearson correlation coefficient of 0.90 via the ImageJ with JaCoP plug-in. (v) Co-localization of live P. gingivalis and F. alocis is also detected in a blood vessel (BV) as denoted with a white arrow. Images were obtained with 20x magnification on Zeiss Axio Imager. SC: Stratum corneum, SL: Stratum lucidum, SG: Stratum granulosum, SB: Stratum basale, LT: Lamina propria. Scale bars = 20µm. (B) A representative three-dimensional view of gingival tissue in periodontitis stained with FISH probes for P. gingivalis and F. alocis to display the spatial information.