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Introduction: The program for mammalian cell growth and division consists of four successive phases; G1, S, G2, and M. Porphyromonas gingivalis may manipulate the host cell cycle to benefit bacterial virulence expression, which likely causes the cell and tissue tropism observed in chronic periodontal infections. We examined P. gingivalis for its effects on cell-cycle modulation in mouse ST2 osteoblastic/stromal cells. Methods: Synchronized ST2 cells were infected with P. gingivalis ATCC33277 (wild-type, WT), gingipain-mutants [KDP136 (Δ rgpAΔ rgpBΔ kgp), KDP129 (Δ rgpAΔ rgpB), and KDP133 (Δ kgp)], and a fimbria-deficient mutant (KDP150) for 24 h, then the cell cycle was evaluated using flow cytometry. Cell-cycle-related molecule expression was examined with a microarray, as well as with quantitative real-time polymerase chain reaction and Western blotting assays. Results: Both the WT and KDP150 strains significantly inhibited cellular proliferation and arrested the cell cycle in the G0/G1 phase, while the expression levels of the cell-cycle regulatory molecules cyclin D and cyclin E were also decreased. In contrast, KDP136 did not show any effects. G1 arrest was also clearly induced by KDP129 and KDP133, with KDP129 being more effective. Conclusion: The present findings suggest that P. gingivalis gingipains reduce cyclin expression and cause early G1 arrest, leading to the inhibition of cellular proliferation. [ABSTRACT FROM AUTHOR]

OBJECTIVE: Tumor necrosis factor (TNF)-alpha is associated with chronic gingival inflammation and reported to induce gingival overgrowth (GO), while phenytoin (PHT) is also known to be a causative agent of GO. We examined the synergistic effect of PHT and TNF-alpha on collagen metabolism in human gingival fibroblasts (HGFs). MATERIALS AND METHODS: HGFs were cultured with TNF-alpha and PHT. Quantitative real-time RT-PCR was employed to determine the mRNA levels for collagen, matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs) and integrin subunits. Cellular collagen endocytosis was determined using a flow-cytometry. RESULTS: The proliferation of HGFs was not affected by TNF-alpha or PHT individually, whereas both synergistically increased collagen accumulation in HGFs. Further, collagen mRNA expression was not increased by TNF-alpha or PHT, although together they markedly prevented cellular collagen endocytosis, associated with the suppression of alpha2beta1-integrin mRNA expression. The mRNA expression of MMP-1 and-2 was suppressed by PHT, while TIMP-1 mRNA expression was enhanced by both TNF-alpha and PHT. CONCLUSION: Our results suggest that TNF-alpha and PHT together cause impaired collagen metabolism by suppression of enzymatic degradation with MMPs/TIMP-1 and integrin-mediated endocytosis. These synergistic effects may also be involved in TNF-alpha- and PHT-induced collagen accumulation, leading to GO. [ABSTRACT FROM AUTHOR]

The fimA gene of Porphyromonas gin givalis, encoding fimbrillin (a subunit protein of fimbriae) has been classified into six genotypes (types I-V and Ib). The genotypic variation was previously suggested to be related to the severity of adult periodontitis in the general population. In this study, we compared inflammatory changes caused by bacterial infection to study pathogenic heterogeneity among the different fimA strains in a mouse abscess model. Bacterial suspensions of 13 P. gingivalis strains representing the six fimA types were subcutaneously injected into female BALB/c mice, and serum sialic acid concentrations were assayed as a quantitative host inflammatory parameter. Type II fimA organisms caused the most significant induction of serum sialic acid, as well as other infectious symptoms, followed by types Ib, IV and V. In contrast, types I and III caused weak inflammatory changes. In addition, fimA mutants of type II strains clearly lost their infectious ability. These findings suggest that fimA genotypic variation affects expression of P. gingivalis virulence. [ABSTRACT FROM AUTHOR]

Bacterial surface structures play a critical role in the initiation of infectious diseases. Various surface components of pathogenic bacteria have been reported to be involved in host injury. There is a great deal of evidence incriminating certain Gram-negative, anaerobic bacteria present in the gingival crevice as etiologic agents of human periodontal diseases. We have isolated endotoxic cellular components from suspected periodontopathic bacteria and examined their immunobiological activities. Lipopolysaccharides (LPS) and lipidassociated proteoglycans (LPG) were prepared from whole cells by the phenol-water and butanol-water procedures, respectively. LPS from Bacteroides gingivalis, B. intermedius, B. oralis, and B. loescheii, Fusobacterium nudeatum and F. necrophorum, and Actinobacillus (Haemophilus) actinomycetemcomitans were found to possess biological activities comparable with those of LPS from E. coli K235 in terms of activation of Limulus lysate, B-cell mitogenicity, polyclonal B-cell activation, induction of bone resorption, and IL-1 production by macrophages. These LPS contained mainly sugars, amino sugars, and fatty acids. No heptose or 2-keto-3-deoxyoctonate (KDO) was detected in the Bacteroides LPS, while LPS from Actinobacillus and Fusobacterium species contained significant amounts of heptose as well as small quantities of KDO. Bacteroides LPS were clearly mitogenic for spleen cells of C3H/HeJ mice, which are non-responsive to LPS from E. coli, A. actinomycetemcomitans, and Fusobacterium species. Furthermore, polymyxin B was found to abrogate the mitogenic activity of LPS from E. coli, Actinobacillus, and Fusobacterium species, but not those from Bacteroides species. Spleen cells from both C3H/HeN and C3H/HeJ mice responded to all butanol-water-extracted LPG preparations, including those from E. coli, A. actinomycetemcomitans, and Fusobacterium species. It may be concluded that LPS and LPG isolated from suspected periodontopathic bacteria possess marked immunobiological potencies on lymphoreticular and bone cells. [ABSTRACT FROM AUTHOR]

The 64-kDa protein to which about half the sera from patients with localized juvenile periodontitis and rapidly progressive periodontitis reacted strongly was purified from Actinobacillus actinomycetemcomitans Y4. Determination of the N-terminal sequence of the protein revealed that it was a GroEL-like protein. The DNA fragment containing the groEL gene of A. actinomycetemcomitans was amplified by polymerase chain reaction, and the groESL operon was cloned by using colony hybridization with the amplified fragment from A. actinomycetemcomitans chromosomal DNA. Sequence analysis revealed that structures of the operon and its products were typical in gram-negative bacteria. Rabbit polyclonal antibodies to the 64-kDa protein cross-reacted with approximately 65-kDa proteins of Haemophilus aphrophilus, Haemophilus influenzae, Haemophilus paraphrophilus, Escherichia coli and Eikenella corrodens but not with any cellular proteins of Porphyromonas gingivalis, Prevotella intermedia and Fusobacteriurn nucleatum. It is possible that antibodies reactive to the 64-kDa protein in periodontitis patients are induced by the cross-reactivity with the hsp60 proteins of other bacteria. [ABSTRACT FROM AUTHOR]

The complete nucleotide sequence of the gene for a cell-surface protein antigen (PAc) of Streptococcus mutans MT8148 (serotype c) was determined. The pac gene consisted of 4695 bp and coded for a 170 773 D protein. The pac gene product contained a putative 38 amino acid signal peptide, resulting in a 166817D mature protein. A potential promoter sequence and a putative Shine­Dalgarno sequence preceded the open reading frame. Two internal repeating amino acid sequences were present in the PAc. One repeating region located in the N-terminal region was rich in alanine, and the other located in the central region was rich in proline. Southern blot analysis under the less stringent condition (allowing up to 35% base mismatch) revealed that the probe covering the prolinerich region hybridized to DNA preparations from strains of Streptococcus cricetus, Streptococcus sobrinus and Streptococcus downei as well as Streptococcus mutans. [ABSTRACT FROM AUTHOR]

The structural gene for a 190kD protein antigen (PAc) of Streptococcus mutans MT8148 (serotype c) was cloned into the plasmid vector pUC118. SDS-polyacrylamide gel electrophoresis and Western immunoblotting showed that the Escherichia coli harbouring the chimaeric plasmid produced multiple polypeptides of 190–210kD. Immunodiffusion analysis revealed that the cloned PAc had the same specific determinants as S. mutans PAc. The cloned pac gene was mapped, and its transcriptional orientation was determined by characterizing deletion mutants of the chimaeric plasmid. Southern blot analysis with the cloned gene sequence as a probe revealed the presence of a homologous sequence in DNAs from serotypes e and f S. mutans. PAc-defective mutants were constructed by inserting an erythromycin-resistance gene into the pac gene. The cell-surface hydrophobicity of the mutants was lower than that of the parent strain. [ABSTRACT FROM AUTHOR]

Lipopolysaccharides (LPS) were isolated from Bacteroides gingivalis, Escherichia coli, Actinobacillus actinomycetemcomitans, Fusobacterium nucleatum and F. necrophorum by the phenol-water procedure. These LPS preparations enhanced the production of prostaglandin E by macrophages from BALB/c and C3H/HeN mice. Only B. gingivalis LPS, however, markedly stimulated the production of prostaglandin E by macrophages from "LPS-nonresponsive" C3H/HeJ mice. [ABSTRACT FROM AUTHOR]