Lipopolysaccharides (LPS) prepared from Bacteroides intermedius (Prevotela intermedia) and Bacteroides (Porphyromonas) gingivalis by hot phenol-water extraction induced interleukin-8 (IL-8) mRNA in normal human gingival fibroblast cultures, as demonstrated by Northern (RNA) blot analysis. IL-8 mRNA levels began to increase after a 2-h exposure, reached a maximum after 12 h, and then dropped to the unstimulated level at 48 h. IL-8 mRNA levels were also enhanced in a dose-dependent manner. By contrast, LPS specimens from various SalmoneUla species with S and R chemotypes and from bacterial and synthetic lipid A preparations did not increase IL-8 mRNA levels in fibroblasts. Although recombinant human IL-la induced IL-8 mRNA expression in fibroblast cultures, an antiserum to recombinant human IL-la did not decrease the IL-8 mRNA accumulation induced by B. intermedius LPS. Fibroblasts primed with natural human gamma interferon (IFN-y) expressed higher IL-8 mRNA levels upon stimulation with B. intermedius LPS, but not with SalmoneUla LPS, compared with nontreated cells. Natural human EFN-j exhibited a similar priming effect on the fibroblasts, and antiserum to IFN-I added to the cultures together with B. intermedius LPS decreased the IL-8 mRNA levels. Therefore, endogenous IFN-I enhanced IL-8 mRNA production in response to B. intermedius LPS in fibroblasts.

Cysteine proteases, including Arg-gingipain of Porphyromonas gingivalis, have been implicated as important virulence factors in periodontal diseases. These enzymes are also involved in the hemagglutinating activity of the organisms. In order to determine the role of proteases in the colonization of the gingival margin, we have compared the attachment properties of P. gingivalis 381 with those of its Arg-gingipain-defective mutant, G-102. Interactions with gram-positive bacteria, human oral epithelial cells, extracellular matrix proteins, and type I collagen were evaluated. In all cases, mutant G-102 was deficient in attachment relative to the parental strain. The mutant's defects could be explained, in part, by the weak autoaggregation displayed by the mutant, which appeared to result from altered fimbrial expression. Both Western blot (immunoblot) and Northern (RNA) blot analyses indicated reduced expression of the major 43-kDa fimbrillin subunit in the mutant. These results suggest that Arg-gingipain may play both direct and indirect roles in the colonization of the gingival margin. In addition, fimbriae may play a direct role in interacting with some host surfaces.

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Large numbers of bacteria coexist in the oral cavity. Streptococcus sanguinis, one of the major bacteria in dental plaque, produces hydrogen peroxide (H 2 O 2 ), which interferes with the growth of other bacteria. Streptococcus mutans, a cariogenic bacterium, can coexist with S. sanguinis in dental plaque, but to do so, it needs a means of detoxifying the H 2 O 2 produced by S. sanguinis. In this study, we investigated the association of three oxidative stress factors, Dpr, superoxide dismutase (SOD), and AhpCF, with the resistance of S. sanguinis to H 2 O 2 . The knockout of dpr and sod significantly increased susceptibility to H 2 O 2 , while the knockout of ahpCF had no apparent effect on susceptibility. In particular, dpr inactivation resulted in hypersensitivity to H 2 O 2 . Next, we sought to identify the factor(s) involved in the regulation of these oxidative stress genes and found that PerR negatively regulated dpr expression. The knockout of perR caused increased dpr expression levels, resulting in low-level susceptibility to H 2 O 2 compared with the wild type. Furthermore, we evaluated the roles of perR, dpr, and sod when S. mutans was cocultured with S. sanguinis. Culturing of the dpr or sod mutant with S. sanguinis showed a significant decrease in the S. mutans population ratio compared with the wild type, while the perR mutant increased the ratio. Our results suggest that dpr and sod in S. mutans are involved in coexistence with S. sanguinis, and PerR is associated with resistance to H 2 O 2 in regulating the expression of Dpr.

Interleukin (IL)-6 expression in human dental pulp cell cultures after stimulation with Prevotella intermedia lipopolysaccharide (LPS) was investigated by Northern blot analysis, enzyme immunoassay, and bioassay. The IL-6 mRNA expression began to increase after 1 hr and continued after up to 8 hr of exposure on stimulation with 10 microg/ml of P. intermedia LPS. The bioactivity was dose-dependent on the concentration of P. intermedia LPS (0 to 100 microg/ml). The IL-6 mRNA expression was inhibited by actinomysin D and super-induced by cycloheximide. Anti-CD14 monoclonal antibody (MY4) inhibited the IL-6 mRNA expression when administered at a 0.5 microg/ml concentration before stimulation with P. intermedia LPS at 1 microg/ml. The immunoregulatory cytokines (interferon-gamma, IL-10, and IL-4) inhibited LPS-induced IL-6 production with a combined treatment. These results suggest the IL-6 expression by pulp cell cultures is CD14-dependent and regulated at the transcriptional level, and a combined treatment with immunoregulatory cytokines may be effective for control of pulpal inflammation due to P. intermedia LPS.

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In order to access the role of the Porphyromonas gingivalis Arg-gingipain proteases in the virulence of this organism, a mutant defective in the rgpA gene was constructed in strain 381. This mutant, MT10, displayed only 40% of the Arg-specific cysteine protease activity of the wild-type strain. In addition, MT10, as well as the recently characterized protease mutant G-102, which is defective in the rgpB gene, displayed reduced self-aggregation, hemagglutination, and the ability to bind to immobilized type I collagen compared to levels of the wild-type parent. However, unlike mutant G-102, the rgpA mutant displayed increased binding to epithelial cells relative to that of the parental organism. Mutant MT10 also did not express detectable levels of the FimA protein as assessed by both Western and Northern blotting or fimbriae visible by electron microscopy of the cells. Furthermore, the ability of MT10 to degrade rat tail collagen fibers when it was cultured at 37°C was markedly attenuated compared to that of strain 381. These results suggest that Arg-gingipain A may play a significant role in the pathogenicity of P. gingivalis by altering the colonization and toxic properties of the organism.

The complete nucleotide sequence of the gene for a cell surface protein antigen (SpaA) of Streptococcus sobrinus MT3791 (serotype g) was determined. The spaA gene consisted of 4,698 bp and coded for a protein of 170,202 Da. A putative signal peptide was found in the amino-terminal end of the protein. A potential promoter sequence and a putative Shine-Dalgarno sequence preceded the open reading frame. Two internal repeating amino acid sequences were present in SpaA. One repeating region, located in the amino-terminal region, was rich in alanine, and the other, located in the central region, was rich in proline. The molecular structure of SpaA was very similar to that of the surface protein antigen of Streptococcus mutans.