Gingipain-dependent Degradation of Intracellular Signalling Proteins by Porphyromonas gingivalis

Methods: Immortalised human oral keratinocytes cells were challenged with P. gingivalis in the presence and absence of cytochalasin D (to block invasion) before total protein extraction.  Levels of members of the mTOR pathway (mTOR, rictor, raptor, Akt and GbetaL) were analysed by immunoblotting.  To probe whether changes observed were mediated by gingipains, an rgp^-kgp^- triple-gingipain mutant was generated and its degradative ability compared to wild-type, rgp^- and kgp^- single-mutants.  In parallel, a cell-free assay employing crude gingipain preparations and keratinocyte lysates was developed to examine the direct involvement of gingipains in vitro.

Results:   Following infection, mTOR was degraded by wild-type P. gingivalis and the rgp^- mutant, but not by the gingipain-free rgp^-kgp^- mutant or the kgp^-single-mutant strain indicating that this degradation is mediated predominantly by Lys-specific gingipains.  This was corroborated in the cell-free assay with the Lys-gingipains.  Degradation was also dependent on invasion as it was inhibited by cytochalasin D.  Changes in several other mTOR-pathway proteins were observed whilst levels of GbetaL remained unchanged.

Conclusions: Our data suggests that P. gingivalis infection causes profound changes in the mTOR signalling pathway, implicating a novel route by which this pathogen may evade innate immune responses.