Infection of the Epithelial Barrier by Porphyromonas gingivalis in vitro

Background: In vitro, oral keratinocytes are able to form an epithelial barrier, which is characterized by a distinct transepithelial electrical resistance (TEER).

P. gingivalis, one of the etiologic agents of periodontal infection, expresses a variety of virulence factors. Primary and immortalised human gingival keratinocytes were used to investigate the influence of P. ginigvalis on the TEER and the critical infectious dose described by the multiplicity of infection (MOI).

Objectives: Analysis of the influence of gingipains and other bacterial proteases on the destruction of the epithelial barrier.

Methods: Keratinocytes were seeded incubated for 24h until they developed a TEER between 80 and 120 Ohmxcm2. Suspensions of different P. gingivalis strains which contained bacteria in a MOI of 101-105 were applied. The specific RGP and KGP gingipain inhibitors FFRck and Z-FKck were added to the bacterial solution. TEER was measured before and at different intervals after infection with the germs. All experiments were repeated twice.

Results: At a MOI 105, immortalised and primary keratinocytes showed a rapid decrease of TEER (49 Ohmxcm2) within the first 24h after infection (p<0.05). In all cases the destruction of the TEER was accelerated (after 12h decrease: 70 Ohmxcm2; p<0.05), if the bacteria were applied to the basolateral part of the insert. Gingipain inhibitors delayed the decrease of the TEER by 12h (at 24h decrease 70 Ohmxcm2; p<0.05). P. gingivalis KDP 136, a gingipain defect mutant (kindly provided by Prof. K. Nakayama, Nagasaki University) similarly demonstrated a delayed destruction of the barrier (at 24h decrease 39 Ohmxcm2; p<0.05).

Conclusions: The impact of P. gingivalis infection on the oral mucosal barrier can be attributed at least in part to the activity of gingipains and other bacterial proteases in this in-vitro model. Primary and immortalised human gingival keratinocytes were shown to react similarly.