Lipopolysaccharide Preparation Extracted fromPorphyromonas gingivalisLipoprotein-Deficient Mutant Shows a Marked Decrease in Toll-Like Receptor 2-Mediated Signaling

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Abstract
We recently demonstrated that a new PG1828-encoded lipoprotein (PG1828LP) was able to be separated from aPorphyromonas gingivalislipopolysaccharide (LPS) preparation, and we found that it exhibited strong cell activation, similar to that ofEscherichia coliLPS, through a Toll-like receptor 2 (TLR2)-dependent pathway. In order to determine the virulence of PG1828LP toward cell activation, we generated a PG1828-deficient mutant ofP. gingivalisstrain 381 by allelic exchange mutagenesis using anermF-ermAMantibiotic resistance cassette. A highly purified preparation of LPS from a PG1828-deficient mutant (ΔPG1828-LPS) showed nearly the same ladder-like patterns in silver-stained gels as a preparation of LPS from a wild-type strain (WT-LPS), as well asLimulusamoebocyte lysate activities that were similar to those of the WT-LPS preparation. However, the ability of the ΔPG1828-LPS preparation to activate NF-κB in TLR2-expressing cells was markedly attenuated. Cytokine production by human gingival fibroblasts was also decreased in response to the ΔPG1828-LPS preparation in comparison with the WT-LPS preparation, and the activity was comparable to the stimulation of highly purified lipid A ofP. gingivalisby TLR4. Further, lethal toxicity was rarely observed following intraperitoneal injection of the PG1828-deficient mutant into mice compared to that with the wild-type strain, while the ΔPG1828-LPS preparation showed no lethal toxicity. Taken together, these results clearly indicate that PG1828LP plays an essential role in inflammatory responses and may be a major virulence factor ofP. gingivalis.