Role of Porphyromonas gingivalis Gingipains in Caspase-1 Activation and Phagocytosis of Tannerella forsythia

Abstract

Objectives In the pathogenesis of periodontitis, Porphyromonas gingivalis plays a role as a keystone pathogen that dysregulates host immune responses and results in dysbiosis in oral microbial communities. Arg-gingipains (RgpA and RgpB) and Lys-gingipain (Kgp) are essential to the virulence of P. gingivalis. Therefore, gingipains are often considered as therapeutic targets. The aim of this study was to elucidate the roles of gingipains in caspase-1 activation in P. gingivalis-infected macrophages and to investigate the roles of gingipains in the modulation by P. gingivalis of phagocytosis of Tannerella forsythia by macrophages.

Methods Macrophages differentiated from THP-1 cells with phorbol-12-myristate-13-acetate (PMA) and peripheral blood mononuclear cells (PBMC)-derived macrophages were infected with P. gingivalis or its gingipain mutants for 6 hours to determine whether P. gingivalis activates caspase-1 and whether gingipain mutation affects the caspase-1 activation. Caspase-1, interleukin (IL)-1β, and lactate dehydrogenase (LDH) in the culture supernatants were analyzed with immunoblot, ELISA, and LDH cytotoxicity assay. To examine the role of gingipain protease activity, macrophages were infected with P. gingivalis in the presence or absence of leupeptin, a cysteine protease inhibitor, or P. gingivalis preincubated with gingipain-specific inhibitors, KYT-1 and KYT-36. Degradation of proteins that were released from cells upon caspase-1 activation was analyzed after incubation of P. gingivalis in the culture supernatants of macrophages that had been stimulated with heat-killed P. gingivalis. Intracellular caspase-1 activity and ATP release were measured after infection with P. gingivalis and its gingipain mutants, or P. gingivalis preincubated with KYT-1 or KYT-36. To assess the effect of processing of surface proteins by gingipains on caspase-1 activation, cells were infected with the gingipain-null mutant that had been cultured overnight with gingipain-containing culture supernatants of P. gingivalis. To determine whether P. gingivalis coinfection has an effect on T. forsythia phagocytosis, PMA-differentiated THP-1 cells and PBMC-derived macrophages were infected with carboxyfluorescein diacetate succinimidyl ester (CFSE)-labelled T. forsythia in the presence or absence of P. gingivalis or its gingipain mutants for 1 hour. Phagocytosis of T. forsythia was analyzed by flow cytometry and confocal microscopy. Coaggregation between the two bacterial species was assessed, and the effect of amino acids that inhibit the coaggregation on T. forsythia phagocytosis was examined. The role of gingipain protease activity was determined using KYT-1 and KYT-36. Intracellular persistence/survival of T. forsythia was analyzed by flow cytometry, confocal microscopy, and 16S rRNA-based viability assay after antibiotic protection.

Results Infection with P. gingivalis at low multiplicity of infections (MOIs), but not at high MOIs, resulted in low levels of IL-1β and LDH without detectable active caspase-1 in the culture supernatants. The proteins released from caspase-1-activated cells were rapidly degraded by gingipains. However, P. gingivalis expressing gingipains induced higher intracellular caspase-1 activity in the infected cells than the gingipain-null mutant. The increased intracellular caspase-1 activity was associated with ATP release from the infected cells. In addition, growing the gingipain-null mutant in the culture supernatants containing gingipains enhanced caspase-1 activation by the mutant. In contrast, inhibition of the protease activity of Kgp or Rgp increased the caspase-1-activating potential of wild-type P. gingivalis, indicating an inhibitory effect of the protease activities of Kgp and Rgp. Phagocytosis of T. forsythia was significantly enhanced by coinfection with P. gingivalis in an MOI- and gingipain-dependent manner. Mutation of either Kgp or Rgp in the coinfecting P. gingivalis resulted in attenuated enhancement of T. forsythia phagocytosis. Inhibition of coaggregation between the two bacterial species reduced phagocytosis of T. forsythia in the mixed infection, and the coaggregation was dependent on gingipains. Inhibition of gingipain protease activities in coinfecting P. gingivalis abated the coaggregation and the enhancement of T. forsythia phagocytosis. However, direct effect of protease activities of gingipains on T. forsythia seemed to be minimal. Although most of the phagocytosed T. forsythia were cleared in infected cells, more T. forsythia remained in cells coinfected with gingipain-expressing P. gingivalis than in cells coinfected with the gingipain-null mutant or infected only with T. forsythia at 24 and 48 hours post-infection.

Conclusion Considering that gingipains are major promising therapeutic targets, it is of great importance to fully characterize the roles of gingipains in the pathogenicity of P. gingivalis. This study demonstrated that gingipains play contradictory roles in caspase-1 activation by P. gingivalis monoinfection, and that gingipains are essential in the augmentation of T. forsythia phagocytosis in the mixed infection. This study provides clues to the role of gingipains in the mechanism by which P. gingivalis dysregulates host immune responses.