The capsule of Porphyromonas gingivalis reduces the immune response of human gingival fibroblasts

Abstract

Background: Periodontitis is a bacterial infection of the periodontal tissues. The Gram-negative anaerobic bacterium Porphyromonas gingivalis is considered a major causative agent. One of the virulence factors of P. gingivalis is capsular polysaccharide (CPS). Non-encapsulated strains have been shown to be less virulent in mouse models than encapsulated strains.

Results: To examine the role of the CPS in host-pathogen interactions we constructed an insertional isogenic P. gingivalis knockout in the epimerase-coding gene epsC that is located at the end of the CPS biosynthesis locus. This mutant was subsequently shown to be non-encapsulated. K1 capsule biosynthesis could be restored by in trans expression of an intact epsC gene. We used the epsC mutant, the W83 wild type strain and the complemented mutant to challenge human gingival fibroblasts to examine the immune response by quantification of IL-1beta, IL-6 and IL-8 transcription levels. For each of the cytokines significantly higher expression levels were found when fibroblasts were challenged with the epsC mutant compared to those challenged with the W83 wild type, ranging from two times higher for IL-1beta to five times higher for IL-8.

Conclusions: These experiments provide the first evidence that P. gingivalis CPS acts as an interface between the pathogen and the host that may reduce the host's pro-inflammatory immune response. The higher virulence of encapsulated strains may be caused by this phenomenon which enables the bacteria to evade the immune system.

Figure 1

Schematic representation of the knockout strategy to construct the epsC insertional mutation in W83. A. The genetic arrangement of the 3'-end of the CPS locus in the W83 wild type strain with the grey rectangles representing the genes present. B. Construct pΔepsC for insertional inactivation of epsC. The 1.2 Kb epsC was inserted into BamHI-EcoRI digested pGEX-6-p3 (oval) and interrupted by insertion of a 1.2 Kb EryF (shaded rectangle) in the single ClaI restriction site present. The dashed lines between A and B show the homologous crossover regions between the plasmid and W83 CPS locus. C. The final arrangement of the 3'-end of the P. gingivalis CPS locus after double crossover showing the insertional inactivation of epsC. Arrows represent the primers used to confirm the integrity of the epsC mutant.

Figure 2

Double immunodiffusion analysis of autoclaved supernatants of P. gingivalis strains. Samples of W83, the epsC mutant and the complemented mutant were tested against the K1-specific antiserum (central well). Note that the white precipitate indicating recognition of CPS with the antiserum is absent in case of the epsC mutant, whereas the intact epsC copy restores the wild-type K1 antiserum recognition in the complemented mutant.

Figure 3

Percoll density gradient centrifugation of W83 and epsC mutant. 1 ml of a OD₆₉₀ = 4 suspension of overnight-grown P. gingivalis was layered on top of a stepwise Percoll gradient (10-80%) and centrifuged at 8000 × g for one hour. The gradient is visualized using fuchsine-stained layers in the marker (M).W83 reproducibly settles in the interfaces of 10-20%, 20-30% and 30-40% where most of the bacterial material is found in the 20-30% interface. The epsC mutant settles as a distinct, granulous band at the 50-60% interface.

Figure 4

Negative capsule staining of fuchsine-stained P. gingivalis cells with India Ink. Phase contrast microscopic picture at a 1000× magnification of (A) W83 wild type strain, (B) epsC mutant and (C) the complemented epsC mutant in an India ink preparation which reveals the capsule as a white halo (arrow). The inset shows an extra six times magnification.

Figure 5

Relative expression of IL-1β, IL-6 and IL-8 genes in human gingival fibroblasts (HGF1) infected with P. gingivalis W83 and the epsC mutant. After a 6-hour challenge with P. gingivalis cells at MOI 1000:1 or 10.000:1 as indicated on the Y-axis, the expression levels of IL-1β, IL-6 and IL-8 in human gingival fibroblasts were measured using RT-PCR and represented as a relative value compared to a non-infected control sample which is set to a value of 1. Significant differences p < 0.01 are indicated by an asterisk.