The oral cavity of healthy individuals contains hundreds of different bacterial, viral, and fungal species. Many of these can associate to form biofilms, which are resistant to mechanical stress or antibiotic treatment. Most are also commensal species, but they can become pathogenic in responses to changes in the environment or other triggers in the oral cavity, including the quality of an individual’s personal hygiene. The complexity of the oral microbiome is being characterized through the newly developed tools of metagenomics. How the microbiome of the oral cavity contributes to health and disease is attracting the interest of a growing number of cell biologists, microbiologists, and immunologists.

Porphyromonas gingivalis, an important periodontal pathogen, infects primary gingival epithelial cells (GECs). Despite the large number of bacteria that replicate inside the GECs, the host cell remains viable. We demonstrate that P. gingivalis triggers rapid and reversible surface phosphatidylserine exposure through a mechanism requiring caspase activation. However, after 1 day of infection, the bacteria no longer induce phosphatidylserine externalization and instead protect infected cells against apoptosis. Infection exerts its effect at the level of mitochondria, as P. gingivalis also blocks depolarization of the mitochondrial transmembrane potential and cytochrome c release. Interestingly, protein kinase B/Akt is phosphorylated during infection, which can be blocked with the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002. Suppression of the PI3K/Akt pathway following staurosporine treatment results in mitochondrial-membrane depolarization, cytochrome c release, DNA fragmentation, and increased apoptosis of infected GECs. Thus, P. gingivalis stimulates early surface exposure of phosphatidylserine, which could downmodulate the inflammatory response, while also promoting host cell survival through the PI3K/Akt pathway.

Summary Interaction between the major fimbriae of Porphyromonas gingivalis and gingival epithelial cells is important for bacterial adhesion and invasion. In this study, we identified integrins as an epithelial cell cognate receptor for P. gingivalis fimbriae. Immunoprecipitation and direct binding assays revealed a physical association between recombinant fimbrillin and β1 integrins. In vitro adhesion and invasion assays demonstrated inhibition of binding and invasion of P. gingivalis by β1 integrin antibodies. In contrast, invasion of a fimbriae‐deficient mutant of P. gingivalis was not affected by integrin antibodies. Infection of gingival epithelial cells with wild‐type P. gingivalis induced tyrosine phosphorylation of the 68 kDa focal adhesion protein paxillin, whereas the fimbriae‐deficient mutant failed to evoke similar changes. Interestingly, activation of paxillin was not accompanied by an increase in the phosphorylation of focal adhesion kinase (FAK). These results provide evidence that P. gingivalis fimbriae promote adhesion to gingival epithelial cells through interaction with β1 integrins, and this association represents a key step in the induction of the invasive process and subsequent cell responses to P. gingivalis infection.

SummaryThe purinergic receptor P2X7 is involved in cell death, inhibition of intracellular infection and secretion of inflammatory cytokines. The role of the P2X7 receptor in bacterial infection has been primarily established in macrophages. Here we show that primary gingival epithelial cells, an important component of the oral innate immune response, also express functional P2X7 and are sensitive to ATP-induced apoptosis. Porphyromonas gingivalis, an intracellular bacterium and successful colonizer of oral tissues, can inhibit gingival epithelial cell apoptosis induced by ATP ligation of P2X7 receptors. A P. gingivalis homologue of nucleoside diphosphate kinase (NDK), an ATP-consuming enzyme, is secreted extracellularly and is required for maximal suppression of apoptosis. An ndk-deficient mutant was unable to prevent ATPinduced host-cell death nor plasma membrane permeabilization in the epithelial cells. Treatment with purified recombinant NDK inhibited ATP-mediated host-cell plasma membrane permeabilization in a dose-dependent manner. Therefore, NDK promotes survival of host cells by hydrolysing extracellular ATP and preventing apoptosis-mediated through P2X 7.

SummaryPorphyromonas gingivalis can inhibit chemically induced apoptosis in primary cultures of gingival epithelial cells through blocking activation of the effector caspase-3. The anti-apoptotic phenotype of P. gingivalis is conserved across strains and does not depend on the presence of fimbriae, as fimbriaedeficient mutants and a naturally occurring nonfimbriated strain were able to impede apoptosis. To dissect the survival pathways modulated by P. gingivalis, protein and gene expression of a number of components of apoptotic death pathways were investigated. P. gingivalis infection of epithelial cells resulted in the phosphorylation of JAK1 and Stat3. Quantitative real-time reverse transcription polymerase chain reaction showed that expression of Survivin and Stat3 itself, targets of activated Stat3, were elevated in P. gingivalis-infected cells. siRNA knockdown of JAK1, in combination with knockdown of Akt, abrogated the ability of P. gingivalis to block apoptosis. In contrast, cIAP-1 and cIAP-2 were not differentially regulated at either the protein or mRNA levels by P. gingivalis. One mechanism by which P. gingivalis can block apoptotic pathways in gingival epithelial cells therefore is through manipulation of the JAK/Stat pathway that controls the intrinsic mitochondrial cell death pathways. Induction of a pro-survival phenotype may prevent programmed host cell death and aid survival of P. gingivalis within gingival epithelial cells.