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Integrins are cell-surface heterodimeric glycoproteins composed of alpha and beta subunits that mediate cell-cell, cell-extracellular matrix, and cell-pathogen interactions. In this study, we report a specific role of integrin α5β1 in NLRP3 inflammasome activation in macrophages stimulated by Td92, a surface protein of the periodontopathogen, Treponema denticola. The direct interaction of Td92 with the cell membrane integrin α5β1 resulted in ATP release and K(+) efflux, which are the main events in NLRP3 activation. This interaction was arginine-glycine-aspartate (RGD)-independent, and Td92 internalization was not required for the activity. An integrin α5β1 antibody and oxATP, an ATP receptor antagonist, inhibited NLRP3 expression, caspase-1 activation, interleukin-1β (IL-1β) secretion, and proIL-1β synthesis, all of which were regulated by NF-κB activation. Therefore, our data has identified the integrin α5β1 as a principal cell membrane receptor for both NLRP3 inflammasome activation and IL-1β transcription by a bacterial protein, which could exaggerate inflammation, a characteristic of periodontitis.

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Oral spirochetes include enormously heterogeneous Treponema species, and some have been implicated in the etiology of periodontitis. In this study, we characterized highly conserved surface proteins in four representative oral spirochetes (Treponema denticola, T. lecithinolyticum, T. maltophilum, and T. socranskii subsp. socranskii) that are homologs of T. pallidum Tp92, with opsonophagocytic potential and protective capacity against syphilis. Tp92 homologs of oral spirochetes had predicted signal peptides (20 to 31 amino acids) and molecular masses of 88 to 92 kDa for mature proteins. They showed amino acid sequence identities of 37.9 to 49.3% and similarities of 54.5 to 66.9% to Tp92. The sequence identities and similarities of Tp92 homologs of oral treponemes to one another were 41.6 to 71.6% and 59.9 to 85.6%, respectively. The tp92 gene homologs were successfully expressed in Escherichia coli, and the recombinant proteins were capable of binding to KB cells, an epithelial cell line, and inhibited the binding of the whole bacteria to the cells. Antiserum (the immunoglobulin G fraction) raised against a recombinant form of the T. denticola Tp92 homolog cross-reacted with homologs from three other species of treponemes. The Tp92 homologs stimulated various factors involved in inflammation and osteoclastogenesis, like interleukin-1␤ (IL-1␤), tumor necrosis factor alpha, IL-6, prostaglandin E 2 , and matrix metalloproteinase 9, in host cells like monocytes and fibroblasts. Our results demonstrate that Tp92 homologs of oral spirochetes are highly conserved and may play an important role in cell attachment, inflammation, and tissue destruction. The coexistence of various Treponema species in a single periodontal pocket and, therefore, the accumulation of multiple Tp92 homologs may amplify the pathological effect in periodontitis.Numerous studies examining epidemiology and virulence factors have revealed strong evidence for the implication of oral spirochetes in the etiology of periodontitis, and the presence of spirochetes in the subgingival plaque is associated with an increased severity of periodontitis (16,38,48). Oral spirochetes include at least 50 phylotypes (12) and account for 20 to

Invasion of periodontal pathogens into periodontal tissues is an important step that can cause tissue destruction in periodontal diseases. Porphyromonas gingivalis is a keystone pathogen and its gingipains are key virulence factors. Fusobacterium nucleatum is a bridge organism that mediates coadhesion of disease-causing late colonizers such as P. gingivalis and early colonizers during the development of dental biofilms. The aim of this study was to investigate how P. gingivalis, in particular its gingipains, influences the invasion of coinfecting F. nucleatum into gingival epithelial cells. When invasion of F. nucleatum was analyzed after 4 h of infection, invasion of F. nucleatum was suppressed in the presence of P. gingivalis compared with during monoinfection. However, coinfection with a gingipain-null mutant of P. gingivalis did not affect invasion of F. nucleatum. Inhibition of PI3K reduced invasion of F. nucleatum. P. gingivalis inactivated the PI3K/AKT pathway, which was also dependent on gingipains. Survival of intracellular F. nucleatum was promoted by P. gingivalis with Arg gingipain mutation. The results suggest that P. gingivalis, in particular its gingipains, can affect the invasion of coinfecting F. nucleatum through modulating intracellular signaling of the host cells.

Caspase-4 is an inflammatory caspase; however, its mechanism of activation is poorly understood. In this study, we demonstrate that Td92, a surface protein of the periodontal pathogen Treponema denticola and a homolog of the Treponema pallidum surface protein Tp92, activates caspase-4 and induces pyroptosis in primary cultured human gingival fibroblasts (HGFs) via cathepsin G activation. Cathepsin G inhibition or siRNA knockdown of cathepsin G inhibited Td92-induced caspase-4 activation and cell death. Td92-induced cell death was significantly inhibited by siRNA knockdown of gasdermin D. Td92 treatment resulted in the binding of cathepsin G to caspase-4 and the coaggregation of these two molecules. In addition, Td92 induced IL-1α expression and secretion, and this was inhibited by caspase-4 knockdown. Cytochalasin D did not block Td92-induced caspase-4 activation, suggesting that Td92 internalization is not required for caspase-4 activation. Our results demonstrate that cathepsin G is directly engaged in caspase-4 activation by a bacterial ligand, which is responsible for cell death and IL-1α secretion in HGFs.

Porphyromonas gingivalis utilizes its major proteases, Arg gingipains (RgpA and RgpB) and Lys gingipain (Kgp), for dysregulation of host immune systems. The aim of this study was to investigate the roles of gingipains in caspase-1 activation and its sequelae in P. gingivalis-infected macrophages. Infection with P. gingivalis at low multiplicity of infections (MOIs), but not at high MOIs, resulted in low levels of interleukin-1β and lactate dehydrogenase 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 with gingipains induced higher intracellular caspase-1 activity in the infected cells than the gingipain-null mutant, which was associated with ATP release from the infected cells. In addition, growing the gingipain-null mutant with gingipains enhanced caspase-1 activation by the mutant. In contrast, inhibition of the protease activity of Kgp or Rgps increased the caspase-1-activating potential of wild-type P. gingivalis, indicating an inhibitory effect of the collaborative action of Kgp and Rgps. These results illuminate the contradictory roles of gingipains in the manipulation of host defence systems by P. gingivalis, as they act by both stimulating and inhibiting innate immune responses.