Mayumi Miura

Humoral Immune Responses to S-Layer-Like Proteins of Bacteroides forsythus

ABSTRACT Bacteroides forsythus is one of the important periodontopathic bacteria, and this microorganism is known to have an S-layer outside the outer membrane. The S-layer-like antigens were recently isolated from B. forsythus, and they were found to be 270- and 230-kDa proteins in the envelope fraction. In this study, these proteins were confirmed to be specific to B. forsythus by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and they were clearly recognized by sera from patients with adult and early-onset periodontitis in Western immmunoblot analysis. We compared the immunoglobulin G (IgG) responses against the purified S-layer-like antigen by enzyme-linked immunosorbent assay. IgG responses against this antigen were low in healthy control subjects, but they were significantly higher in subjects with adult and early-onset periodontitis. Together with the fact that the IgG responses against the crude extract of B. forsythus did not rise significantly in patients with periodontitis, S-layer-like proteins are considered to be specific antigens of B. forsythus and may play an important role in the progression of periodontitis.

Regenerative effect of azithromycin on periodontitis with different levels of gingival inflammation: three case reports

BACKGROUND Azithromycin is an antibiotic belonging to the macrolides. Previous case reports showed that azithromycin has a regenerative effect on periodontal tissue in addition to improving periodontal gingival inflammation. Recently, we experienced three periodontitis cases, all of which showed severe bone loss. However, their gingival inflammatory signs differed greatly. The present case reports evaluated the regenerative effects of azithromycin on periodontitis sites with different clinical signs of gingival inflammation. METHODS In Case 1, generalized chronic periodontitis with severe gingival inflammation was treated with azithromycin before periodontal treatment. In contrast, Case 2 presented with few clinical signs of gingival inflammation, but was treated with azithromycin prescribed within a day of scaling and root planing. In Case 3, teeth with moderate gingival inflammation were treated with azithromycin after a series of scaling and root planing. RESULTS Remarkable alveolar bone growth, regardless of baseline gingival inflammation, was noted in all three cases. CONCLUSIONS The use of adjunctive azithromycin in scaling and root planing may be effective for periodontal tissue regeneration. This property may be independent of the degree of baseline gingival inflammation.

Serotype-dependent expression patterns of stabilized lipopolysaccharide aggregates in Aggregatibacter actinomycetemcomitans strains.

Above a critical concentration, amphiphilic lipopolysaccharide (LPS) molecules in an aqueous environment form aggregate structures, probably because of interactions involving hydrophobic bonds. Ionic bonds involving divalent cations stabilize these aggregate structures, making them resistant to breakdown by detergents. The aim of this study was to examine expression patterns of stabilized LPS aggregates in Aggregatibacter actinomycetemcomitans, a microorganism that causes periodontitis. A. actinomycetemcomitans strains of various serotypes and truncated LPS mutants were prepared for this study. Following treatment with a two‐phase separation system using the detergent Triton X‐114, crude LPS extracts of the study strains were separated into detergent‐phase LPS (DP‐LPS) and aqueous‐phase LPS (AP‐LPS). Repeated treatment of the aqueous phase with the two‐phase separation system produced only a slight decrease in AP‐LPS, suggesting that AP‐LPS was resistant to the detergent and thus distinguishable from DP‐LPS. The presence of divalent cations increased the yield of AP‐LPS. AP‐LPS expression patterns were serotype‐dependent; serotypes b and f showing early expression, and serotypes a and c late expression. In addition, highly truncated LPS from a waaD (rfaD) mutant were unable to generate AP‐LPS, suggesting involvement of the LPS structure in the generation of AP‐LPS. The two‐phase separation was able to distinguish two types of LPS with different physical states at the supramolecular structure level. Hence, AP‐LPS likely represents stabilized LPS aggregates, whereas DP‐LPS might be derived from non‐stabilized aggregates. Furthermore, time‐dependent expression of stabilized LPS aggregates was found to be serotype‐dependent in A. actinomycetemcomitans.

A novel gene required for natural competence in Aggregatibacter actinomycetemcomitans

BACKGROUND AND OBJECTIVE Natural competence is the ability of bacteria to take up extracellular DNA and incorporate it into their genomes. Some strains of Aggregatibacter actinomycetemcomitans, a critical periodontal pathogen, are naturally competent for transformation. However, information on natural competence genes is limited for this species. The aim of this study was to confirm the involvement of a novel gene identified near the fimbriae gene cluster in natural competence. MATERIAL AND METHODS The functions of putative open reading frames (ORFs), designated AA00863-AA00865, in the Oralgen project database for A. actinomycetemcomitans strain HK1651, have not been determined. Using naturally transformable A. actinomycetemcomitans strains D7S-1 and ATCC29523, we created deletion mutants of homologous genes of these ORFs. Natural competence in the study strains was determined using an agar-based transformation frequency assay. RESULTS Mutation of the AA00865 homolog, which we named urpA in A. actinomycetemcomitans strain D7S-1, resulted in the loss of natural competence, whereas mutations of the AA00864 and AA00863 homologs, located downstream of urpA gene, did not. Similar results were also observed in the mutants of A. actinomycetemcomitans ATCC29523. Complementation of the deleted sequence in the urpA mutant restored natural competence. CONCLUSION The urpA gene is a novel gene required for natural competence in A. actinomycetemcomitans and does not exhibit significant homology with any natural competence genes previously identified in other bacterial species.

Antibiotic effects against periodontal bacteria in organ cultured tissue

Mechanical reduction of infectious bacteria by using physical instruments is considered the principal therapeutic strategy for periodontal disease; addition of antibiotics is adjunctive. However, local antibiotic treatment, combined with conventional mechanical debridement, has recently been shown to be more effective in periodontitis subjects with type 2 diabetes. This suggests that some bacteria may invade the inflamed inner gingival epithelium, and mechanical debridement alone will be unable to reduce these bacteria completely. Therefore, we tried to establish infected organ culture models that mimic the inner gingival epithelium and aimed to see the effects of antibiotics in these established models. Mouse dorsal skin epithelia were isolated, and periodontal bacteria were injected into the epithelia. Infected epithelia were incubated with test antibiotics, and colony‐forming ability was evaluated. Results indicated that effective antibiotics differed according to injected bacteria and the bacterial combinations tested. Overall, in organ culture model, the combination of amoxicillin or cefdinir and metronidazole compensate for the effects of less effective bacterial combinations on each other. This in vitro study would suggest effective periodontal treatment regimens, especially for severe periodontitis.