In vitro models of tissue penetration and destruction by Porphyromonas gingivalis

Abstract

Porphyromonas gingivalis is a gram-negative anaerobic bacterium that is considered the key etiologic agent of chronic periodontitis. Arg- and Lys-gingipain cysteine proteinases produced by P. gingivalis are key virulence factors and are believed to be essential for significant tissue component degradation, leading to host tissue invasion by periodontopathogens. Two in vitro models were used to determine the extent to which P. gingivalis can reach connective tissue. The tissue penetration potential of P. gingivalis was first investigated by using an engineered human oral mucosa model composed of normal human epithelial cells and fibroblasts. Internalized bacteria were assessed by transmission electron microscopy. Bacteria were observed within multilayered gingival epithelial cells and in the space between the stratified epithelium and the lamina propria. A gingipain-null mutant strain of P. gingivalis was found to be less potent in penetrating tissue than the wild-type strain. Proinflammatory responses to P. gingivalis infection were evaluated. P. gingivalis increased the secretion of interleukin-1 beta, interleukin-6, interleukin-8, and tumor necrosis factor alpha. In the second part of the study, the contribution of P. gingivalis gingipains to tissue penetration was investigated by using a reconstituted basement membrane model (Matrigel). The penetration of (14)C-labeled P. gingivalis cells through Matrigel was significantly reduced when leupeptin, a specific inhibitor of Arg-gingipain activity, was added or when a gingipain-null mutant was used. The results obtained with these two relevant models support the capacities of P. gingivalis to infiltrate periodontal tissue and to modulate the proinflammatory response and suggest a critical role of gingipains in tissue destruction.

FIG. 1.

Effect of P. gingivalis on EHOM structures revealed by optical microscopy. (a and b) Histological structures of sections of the control EHOM placed in a CO₂ (a) or an anaerobic (b) atmosphere for 24 h. (c and d) Histological structures of sections of the EHOM infected with P. gingivalis ATCC 33277 (10⁹ cells) (c) or P. gingivalis KDP128 (10⁹ cells) (d) for 24 h. Biopsy specimens were collected and embedded in paraffin. Thin sections were stained with Masson trichome. Scale bars, 50 μm.

FIG.2.

Effect of P. gingivalis on EHOM structures revealed by transmission electron microscopy. Shown are ultrathin sections of the EHOM infected with P. gingivalis ATCC 33277 (10⁹ cells) or P. gingivalis KDP128 (10⁹ cells). Cells of P. gingivalis ATCC 33277 were localized in multilayered epithelial cells (A) as well as in connective tissue (C), whereas cells of P. gingivalis KDP128 (B) were localized only in multilayered epithelial cells.

FIG. 3.

Effect of P. gingivalis infection on IL-1β, IL-6, IL-8, and TNF-α secretion by the EHOM model. After 24 h of infection, culture supernatants were collected from EHOM samples, and cytokine levels were measured with ELISA kits. Results are reported as the means and SDs of three different EHOM experiments per condition. Single and double asterisks indicate P values of <0.05 and <0.01, respectively, for comparisons of infected and uninfected tissues.

FIG. 4.

Comparative analysis of the penetration potential of P. gingivalis ATCC 33277 and its gingipain-null derivative mutant (KDP128) in the reconstituted basement membrane (Matrigel) model. ¹⁴C-labeled P. gingivalis cells were placed on top of the Matrigel, and incubation was carried out for 24 or 48 h. When the effect of proteinase inhibitors was tested, bacterial cells were preincubated in the presence of the inhibitors prior to initiation of the penetration assays. Radioactivity in the lower chamber was measured with a scintillation counter. The values represent the percent recovery of initial radioactivity added to the upper chamber and are reported as the means and SDs of nine assays. Single, double, and triple asterisks indicate significant differences at P values of <0.05, <0.01, and <0.002, respectively.

FIG. 5.

Degradation of Matrigel constituents by cells of P. gingivalis ATCC 33277 and KDP128, as determined by SDS-PAGE analysis and Coomassie blue staining. (A) Lanes: 1, molecular weight markers; 2, Matrigel alone; 3, Matrigel plus ATCC 33277; 4, Matrigel plus ATCC 33277 and leupeptin; 5, Matrigel plus ATCC 33277 and cathepsin B inhibitor II; 6, Matrigel plus KDP128 (rgpA rgpB kgp). (B) The relative intensities of bands were assessed by using Scion Imaging Software. A value of 1 was given to each band of the Matrigel control. Results are reported as means and SDs.