PMC

P. gingivalis ATCC 33277 and F. nucleatum ATCC 25586 (Fn) were injected into subcutaneously implanted chambers in mice, either alone (10⁹ CFU) or together (5×10⁸ CFU each), and 24h postinfection chamber fluid was aspirated to determine viable CFU counts. The data are means ± SD (n = 5 mice). *, p < 0.01 between Fn and Pg CFU when inoculated alone; •, p < 0.01 between CFU counts of the same organism recovered from single infections and co-infection.

(A) Confocal colocalization of P. gingivalis (green), C5aR (red), and TLR2 (blue). Bottom right, merged image. (B) Fluorescence resonance energy transfer between the indicated donors and acceptors measured from the increase in donor (Cy3 or FITC) fluorescence after acceptor (Cy5 or TRITC) photobleaching. Data are means ± SD (n=3). *, p < 0.01 between the indicated groups and controls. FRET analysis has revealed significant energy transfer between Cy3-labeled C5aR and Cy5-labeled TLR2 in P. gingivalis-stimulated but not resting macrophages, whereas no significant energy transfer was detected between Cy3-labeled C5aR and Cy5-labeled TLR5 or MHC Class I (controls). Moreover, significant energy transfer was observed between FITC-labeled P. gingivalis and TRITC-labeled C5aR or TLR2 (but not TLR5 or MHC Class I). C5aR appeared to associate with P. gingivalis in a TLR2-dependent way since the P. gingivalis-C5aR FRET association was abrogated in Tlr2^−/− macrophages. (The data are from the reference ) (Used by permission).

In macrophages, P. gingivalis interacts with the CD14/TLR2/TLR1 receptor complex (). Moreover, P. gingivalis uses its gingipains to attack C5 and release biologically C5a (, ). Upon C5aR binding, C5a stimulates Gαi-dependent intracellular Ca²⁺ signaling which synergistically enhances the otherwise weak cAMP responses induced by TLR2/TLR1 activation alone. Maximal cAMP induction is achieved by the participation of another G protein-coupled receptor, the CXCR4, which interacts directly with P. gingivalis and coassociates with both TLR2 and C5aR in lipid rafts (, ). The ensuing activation of the cAMP-dependent protein kinase A (PKA) pathway inactivates glycogen synthase kinase-3β (GSK3β) and impairs the inducible nitrogen synthase (iNOS)-dependent killing of the pathogen in macrophages in vitro and in vivo (). The interaction of P. gingivalis with TLR2/TLR1 also activates inside-out signaling, propagated by Rac1, PI3K, and cytohesin-1 (Cyt-1), which induces the high-affinity conformation of CR3 (, ). CR3 then binds and internalizes P. gingivalis; this is a relatively safe portal of entry since CR3 is not linked to vigorous microbicidal mechanisms (, ). The CR3-P. gingivalis interaction also leads to induction of ERK1/2 signaling. This in turn downregulates IL-12 p35 and p40 mRNA expression (), possibly through suppression of a critical transcription factor (the interferon regulatory factor 1 [IRF1]) that is required for IL-12 expression (). This inhibitory ERK1/2 pathway is also activated downstream of the C5aR. The suppressive effects of CR3 and C5aR on TLR2-induced cytokine production are selective for IL-12 and do not affect induction of other proinflammatory cytokines (e.g., IL-1β, IL-6, and TNF-α) in vitro or in vivo. Inhibition of bioactive IL-12 by these mechanisms results in impaired immune clearance of P. gingivalis in vivo (, ). P. gingivalis interacts with at least one more TLR, the TLR4, which, however, is proactively antagonized by an atypical lipopolysaccharide lipid A moiety ().