Porphyromonas gingivalis is the primary etiologic agent of periodontal disease that is associated with other human chronic inflammatory diseases, including atherosclerosis. The ability of P. gingivalis to invade and persist within human aortic endothelial cells (HAEC) has been postulated to contribute to a low to moderate chronic state of inflammation, although how this is specifically achieved has not been well defined. In this study, we demonstrate that P. gingivalis infection of HAEC resulted in the rapid cleavage of receptor interacting protein 1 (RIPK1), a mediator of tumor necrosis factor (TNF) receptor-1 (TNF-R1)-induced cell activation or death, and RIPK2, a key mediator of both innate immune signaling and adaptive immunity. The cleavage of RIPK1 or RIPK2 was not observed in cells treated with apoptotic stimuli, or cells stimulated with agonists to TNF-R1, nucleotide oligomerization domain receptor 1(NOD1), NOD2, Toll-like receptor 2 (TLR2) or TLR4. P. gingivalis-induced cleavage of RIPK1 and RIPK2 was inhibited in the presence of a lysine-specific gingipain (Kgp) inhibitor. RIPK1 and RIPK2 cleavage was not observed in HAEC treated with an isogenic mutant deficient in the lysine-specific gingipain, confirming a role for Kgp in the cleavage of RIPK1 and RIPK2. Similar proteolysis of poly (ADP-ribose) polymerase (PARP) was observed. We also demonstrated direct proteolysis of RIPK2 by P. gingivalis in a cell-free system which was abrogated in the presence of a Kgp-specific protease inhibitor. Our studies thus reveal an important role for pathogen-mediated modification of cellular kinases as a potential strategy for bacterial persistence within target host cells, which is associated with low-grade chronic inflammation, a hallmark of pathogen-mediated chronic inflammatory disorders.
A number of successful pathogens have evolved mechanisms to evade host defenses, thus establishing persistent and chronic infections. Although membrane-bound innate immune receptors including Toll-like receptors play a role in inflammation in response to the common oral pathogen Porphyromonas gingivalis, it is not known how intracellular host defense mechanisms to this pathogen contribute to persistent infection and resulting chronic inflammation. In this study, we have defined a novel strategy by which P. gingivalis modulates the levels of key intracellular proteins involved in cell death and host defense responses. We demonstrate that the lysine-specific bacterial cysteine protease of P. gingivalis (Kgp) induces the proteolysis of receptor interacting protein kinase 1 (RIPK1), RIPK2 and poly (ADP-ribose) polymerase (PARP). Although endogenous host mechanisms may contribute to this process, activation of innate immune signaling cascades, caspases, or apoptosis alone were not sufficient to drive proteolysis. These findings support a role for pathogen-mediated modification of cellular kinases as a strategy for bacterial persistence within target host cells. Together with other recently described mechanisms for P. gingivalis host immune evasion, our work supports the emerging concept that pathogen-mediated chronic inflammatory disorders result from specific pathogen-mediated evasion strategies resulting in low-grade chronic inflammation.