Real-Time TaqMan PCR for Quantifying Oral Bacteria during Biofilm Formation

A TaqMan PCR was developed for quantifying early colonizer microorganisms in dental biofilms. To design species-specific primers and TaqMan probes, genomic subtractive hybridization was used. This quantitative assay in combination with subtractive hybridization may be of value in the study of microbial ecosystems consisting of related species that are involved in the formation and etiology of biofilms

Discrimination of Streptococcus pneumoniae from Viridans Group Streptococci by Genomic Subtractive Hybridization

Two oligonucleotide primer sets for the discrimination of Streptococcus pneumoniae from “pneumococcus-like” oral streptococcal isolates by PCR were developed. Genomic subtractive hybridization was performed to search for differences between Streptococcus pneumoniae strain WU2 and the most closely related oral streptococcus, Streptococcus mitis strain 903. We identified 19 clones that contained S. pneumoniae-specific nucleotide fragments that were absent from the chromosomal DNA of typical laboratory strains of S. mitis and other oral bacteria. Subsequently, oligonucleotide PCR primers for the detection of S. pneumoniae were designed from the sequences of the subtracted DNA fragments, and the specificities of the 19 primer sets were evaluated by PCR using chromosomal DNAs extracted from four S. pneumoniae clinical isolates and from 20 atypical organisms classified as S. mitis or S. oralis, which harbored genes encoding the pneumococcal virulence factors autolysin (lytA) or pneumolysin (ply), as templates. Of the 19 primer sets, two (Spn9802 and Spn9828) did not amplify PCR products from any of the pneumococcus-like streptococcal strains that we examined. The genes containing the Spn9802 and Spn9828 sequences encoded proteins of unknown function that did not correspond to any previously described proteins in other bacteria. These new oligonucleotide primers may be very useful for early and correct diagnosis of S. pneumoniae infections

<i>Porphyromonas gingivalis</i> Gingipain-Dependently Enhances IL-33 Production in Human Gingival Epithelial Cells

<div><p>The cytokine IL-33 is constitutively expressed in epithelial cells and it augments Th2 cytokine-mediated inflammatory responses by regulating innate immune cells. We aimed to determine the role of the periodontal pathogen, <i>Porphyromonas gingivalis</i>, in the enhanced expression of IL-33 in human gingival epithelial cells. We detected IL-33 in inflamed gingival epithelium from patients with chronic periodontitis, and found that <i>P</i>. <i>gingivalis</i> increased IL-33 expression in the cytoplasm of human gingival epithelial cells <i>in vitro</i>. In contrast, lipopolysaccharide, lipopeptide, and fimbriae derived from <i>P</i>. <i>gingivalis</i> did not increase IL-33 expression. Specific inhibitors of <i>P</i>. <i>gingivalis</i> proteases (gingipains) suppressed IL-33 mRNA induction by <i>P</i>. <i>gingivalis</i> and the <i>P</i>. <i>gingivalis</i> gingipain-null mutant KDP136 did not induce IL-33 expression. A small interfering RNA for protease-activated receptor-2 (PAR-2) as well as inhibitors of phospholipase C, p38 and NF-κB inhibited the expression of IL-33 induced by <i>P</i>. <i>gingivalis</i>. These results indicate that the PAR-2/IL-33 axis is promoted by <i>P</i>. <i>gingivalis</i> infection in human gingival epithelial cells through a gingipain-dependent mechanism.</p></div

Induction of IL-33 mRNA expression by <i>P</i>. <i>gingivalis</i> requires activation of the p38 pathway.

<p>(A) Ca9-22 cells stimulated for indicated periods with 50 μg/ml of whole <i>P</i>. <i>gingivalis</i> W83 cells in medium containing 1% FBS. Phosphorylation of p38 was detected in cell lysates by Western blotting against anti-phospho-p38 antibody (p-p38). Controls comprised antibody against total p38. Data are representative of three independent experiments. Relative expression of phosphorylated p38 was quantified using densitometry. Relative expression of phosphorylated p38 was normalized to that of p38. (B) Ca9-22 cells incubated with 10 μM PD98059, SP600125, or SB203580 for 30 min and then stimulated with 50 μg/ml of whole <i>P</i>. <i>gingivalis</i> W83 cells for 48 h in medium containing 5% FBS. Total cellular RNA was extracted and transcripts were analyzed by RT-qPCR. Data are representative of three independent experiments, and are shown as means ± SD of triplicate assays. Statistical significant differences are indicated (*, <i>P</i><0.05 compared with untreated control; §, <i>P</i><0.05 compared with <i>P</i>. <i>gingivalis</i> alone). (C) Whole <i>P</i>. <i>gingivalis</i> W83 cells (50 μg/ml) were incubated with 0.3 μM FPR-cmk plus 0.3 μM KYT-36 for 15 min at 37°C and then used to stimulate Ca9-22 cells for 18 h in medium containing 1% FBS. (D) Ca9-22 cells were stimulated with 50 μg/ml of <i>P</i>. <i>gingivalis</i> ATCC 33277 wild-type or KDP 136 whole cells for 18 h. Cell lysates were analyzed by Western blotting using anti-phospho-p38 and anti-p38 antibodies. Data are representative of three independent experiments. Relative expression levels of phosphorylated p38 were normalized to levels of p38 and quantified by densitometry. Controls were adjusted to contain 0.1% (v/v) DMSO in media during incubation with inhibitors.</p

Induction of IL-33 mRNA expression by <i>P</i>. <i>gingivalis</i> requires activation of the NF-κB pathway.

<p>(A) Ca9-22 cells stimulated with 50 μg/ml of whole <i>P</i>. <i>gingivalis</i> W83 cells for indicated periods. (B) Whole <i>P</i>. <i>gingivalis</i> W83 cells (50 μg/ml) were incubated with gingipain inhibitors (0.3 μM FPR-cmk plus 0.3 μM KYT-36) for 15 min at 37°C, and then used to stimulate Ca9-22 cells for 9 h. (C) Ca9-22 cells stimulated with 50 μg/ml of whole <i>P</i>. <i>gingivalis</i> ATCC 33277 wild-type or KDP136 cells for 9 h. (A-C) Cells were transiently transfected with pNFκB-<i>Metridia</i> luciferase reporter or control p<i>Metridia</i> luciferase reporter plasmids. Amount of secreted luciferase in culture supernatants were analyzed using a luminometer. (D) Ca9-22 cells incubated with indicated concentrations of PDTC (NF-κB inhibitor) for 1 h and then stimulated with 50 μg/ml of whole <i>P</i>. <i>gingivalis</i> W83 cells for 48 h. Total cellular RNA was extracted and transcripts were analyzed by RT-qPCR. Data are representative of three independent experiments and are shown as means ± SD of triplicate assays. Statistical significant differences are indicated (*, <i>P</i><0.05 compared with respective unstimulated control; §, <i>P</i><0.05 compared with <i>P</i>. <i>gingivalis</i> alone). (E) Ca9-22 cells were incubated with 25 μM PCTC for 1 h and then stimulated for 18 h with 50 μg/ml of whole <i>P</i>. <i>gingivalis</i> W83 cells in medium containing 1% FBS. Cell lysates were analyzed by Western blotting against anti-phospho-p38 and anti-p38 antibodies. Data are representative of three independent experiments. Relative expression levels of phosphorylated p38 were normalized to p38 and quantified by densitometry. Control media was adjusted to contain 0.1% (v/v) DMSO during incubation with inhibitor.</p

<i>P</i>. <i>gingivalis</i> increases IL-33 protein expression in gingival epithelial cells.

<p>(A) Ca9-22 cells were stimulated with 50 μg/ml of whole <i>P</i>. <i>gingivalis</i> W83 cells for the indicated periods. Cell lysates were analyzed by Western blotting with an anti-human IL-33 mAb. Expression levels of IL-33 were quantified by densitometry using ImageJ software and normalized to medium alone. (B) Ca9-22 cells were stimulated with 50 μg/ml of whole <i>P</i>. <i>gingivalis</i> W83 cells for 4 d, and intracellular IL-33 protein was stained with PE-conjugated anti-human IL-33 mAb. Nuclei were stained with DAPI. Bar = 25 μm. Data are representative of three independent experiments.</p