Hiroaki Inaba

Detection of oral bacteria in cardiovascular specimens.

BACKGROUND/AIMS Oral bacteria, including cariogenic and periodontal pathogens, are thought to be etiological factors in the development of cardiovascular diseases. To define this relationship, we analyzed the distribution of oral bacterial species in cardiovascular specimens. METHOD Following acceptance into the study, 203 consecutive patients were analyzed, from whom 82 aortic valve specimens, 35 mitral valve specimens, and 86 aortic aneurysmal wall specimens, of which 16 contained aneurysmal thrombus tissues, were obtained. In addition, a total of 58 dental plaque specimens were collected from the same group of patients who underwent heart valve replacement or removal of aortic aneurysms. Bacterial DNA was extracted from both cardiovascular tissues and dental plaque in those cases and then species-specific polymerase chain reaction assays were used to analyze the occurrences of six oral streptococcal and six periodontal bacterial species. RESULTS Streptococcus mutans was the most frequently detected species in the cardiovascular specimens, followed by Aggregatibacter actinomycetemcomitans. As for dental plaque specimens from patients who underwent cardiovascular operations, most of the tested periodontitis-related species as well as oral streptococci were detected at high frequencies. Furthermore, the positive rate of S. mutans in cardiovascular specimens from patients whose dental plaque specimens were also positive for S. mutans was 78%, which was significantly higher than any other tested species when the same analysis was performed. CONCLUSION Our results suggest that specific oral bacterial species, such as S. mutans and A. actinomycetemcomitans, are related to bacteremia and may be etiologic factors for the development of cardiovascular diseases.

Virulence of Porphyromonas gingivalis is altered by substitution of fimbria gene with different genotype.

Porphyromonas gingivalis is a periodontal pathogen whose fimbriae are classified into six genotypes based on the diversity of the fimA genes encoding each fimbria subunit. It was suggested that P. gingivalis strains with type II fimbriae were more virulent than type I strains. For the present study, we generated the mutants in which fimA was substituted with different genotypes to study virulence of type II fimbriae. Using plasmid vectors, fimA of ATCC33277 (type I strain) was substituted with type II fimA, and that of OMZ314 (type II strain) with type I fimA. The substitution of type I fimA with type II enhanced bacterial adhesion/invasion to epithelial cells, whereas substitution with type I fimA resulted in diminished efficiency. Following bacterial invasion, type II clones swiftly degraded cellular paxillin and focal adhesion kinase, and inhibited cellular migration, whereas type I clones and ΔfimA mutants did not. BIAcore analysis demonstrated that type II fimbriae possess greater adhesive abilities for their receptor α5β1‐integrin than those of type I. In a mouse abscess model, the type II clones significantly induced serum IL‐1β and IL‐6, as well as other infectious symptoms. These results suggest that type II fimbriae are a critical determinant of P. gingivalis virulence.

Porphyromonas gingivalis Induces Receptor Activator of NF-κB Ligand Expression in Osteoblasts through the Activator Protein 1 Pathway

ABSTRACT Porphyromonas gingivalis, an important periodontal pathogen, is closely associated with inflammatory alveolar bone resorption, and several components of the organism such as lipopolysaccharides have been reported to stimulate production of cytokines that promote inflammatory bone destruction. We investigated the effect of infection with viable P. gingivalis on cytokine production by osteoblasts. Reverse transcription-PCR and real-time PCR analyses revealed that infection with P. gingivalis induced receptor activator of nuclear factor κB (NF-κB) ligand (RANKL) mRNA expression in mouse primary osteoblasts. Production of interleukin-6 was also stimulated; however, osteoprotegerin was not. SB20350 (an inhibitor of p38 mitogen-activated protein kinase), PD98059 (an inhibitor of classic mitogen-activated protein kinase kinase, MEK1/2), wortmannin (an inhibitor of phosphatidylinositol 3 kinase), and carbobenzoxyl-leucinyl-leucinyl-leucinal (an inhibitor of NF-κB) did not prevent the RANKL expression induced by P. gingivalis. Degradation of inhibitor of NF-κB-alpha was not detectable; however, curcumin, an inhibitor of activator protein 1 (AP-1), prevented the RANKL production induced by P. gingivalis infection. Western blot analysis revealed that phosphorylation of c-Jun, a component of AP-1, occurred in the infected cells, and an analysis of c-Fos binding to an oligonucleotide containing an AP-1 consensus site also demonstrated AP-1 activation in infected osteoblasts. Infection with P. gingivalis KDP136, an isogenic deficient mutant of arginine- and lysine-specific cysteine proteinases, did not stimulate RANKL production. These results suggest that P. gingivalis infection induces RANKL expression in osteoblasts through AP-1 signaling pathways and cysteine proteases of the organism are involved in RANKL production.

Distinct roles of long/short fimbriae and gingipains in homotypic biofilm development by Porphyromonas gingivalis.

BackgroundPorphyromonas gingivalis, a periodontal pathogen, expresses a number of virulence factors, including long (FimA) and short (Mfa) fimbriae as well as gingipains comprised of arginine-specific (Rgp) and lysine-specific (Kgp) cysteine proteinases. The aim of this study was to examine the roles of these components in homotypic biofilm development by P. gingivalis, as well as in accumulation of exopolysaccharide in biofilms.ResultsBiofilms were formed on saliva-coated glass surfaces in PBS or diluted trypticase soy broth (dTSB). Microscopic observation showed that the wild type strain formed biofilms with a dense basal monolayer and dispersed microcolonies in both PBS and dTSB. A FimA deficient mutant formed patchy and small microcolonies in PBS, but the organisms proliferated and formed a cohesive biofilm with dense exopolysaccharides in dTSB. A Mfa mutant developed tall and large microcolonies in PBS as well as dTSB. A Kgp mutant formed markedly thick biofilms filled with large clumped colonies under both conditions. A RgpA/B double mutant developed channel-like biofilms with fibrillar and tall microcolonies in PBS. When this mutant was studied in dTSB, there was an increase in the number of peaks and the morphology changed to taller and loosely packed biofilms. In addition, deletion of FimA reduced the autoaggregation efficiency, whereas autoaggregation was significantly increased in the Kgp and Mfa mutants, with a clear association with alteration of biofilm structures under the non-proliferation condition. In contrast, this association was not observed in the Rgp-null mutants.ConclusionThese results suggested that the FimA fimbriae promote initial biofilm formation but exert a restraining regulation on biofilm maturation, whereas Mfa and Kgp have suppressive and regulatory roles during biofilm development. Rgp controlled microcolony morphology and biovolume. Collectively, these molecules seem to act coordinately to regulate the development of mature P. gingivalis biofilms.

Invasion of epithelial cells and proteolysis of cellular focal adhesion components by distinct types of Porphyromonas gingivalis fimbriae

ABSTRACT Porphyromonas gingivalis fimbriae are classified into six types (types I to V and Ib) based on the fimA genes encoding FimA (a subunit of fimbriae), and they play a critical role in bacterial interactions with host tissues. In this study, we compared the efficiencies of P. gingivalis strains with distinct types of fimbriae for invasion of epithelial cells and for degradation of cellular focal adhesion components, paxillin, and focal adhesion kinase (FAK). Six representative strains with the different types of fimbriae were tested, and P. gingivalis with type II fimbriae (type II P. gingivalis) adhered to and invaded epithelial cells at significantly greater levels than the other strains. There were negligible differences in gingipain activities among the six strains; however, type II P. gingivalis apparently degraded intracellular paxillin in association with a loss of phosphorylation 30 min after infection. Degradation was blocked with cytochalasin D or in mutants with fimA disrupted. Paxillin was degraded by the mutant with Lys-gingipain disrupted, and this degradation was prevented by inhibition of Arg-gingipain activity by Nα-p-tosyl-l-lysine chloromethyl ketone. FAK was also degraded by type II P. gingivalis. Cellular focal adhesions with green fluorescent protein-paxillin macroaggregates were clearly destroyed, and this was associated with cellular morphological changes and microtubule disassembly. In an in vitro wound closure assay, type II P. gingivalis significantly inhibited cellular migration and proliferation compared to the cellular migration and proliferation observed with the other types. These results suggest that type II P. gingivalis efficiently invades epithelial cells and degrades focal adhesion components with Arg-gingipain, which results in cellular impairment during wound healing and periodontal tissue regeneration.

Distribution of Porphyromonas gingivalis fimA genotypes in cardiovascular specimens from Japanese patients.

INTRODUCTION Porphyromonas gingivalis, a major periodontal pathogen, is gaining increasing attention for its possible association with cardiovascular diseases. Its fimbriae are classified into six genotypes (types I-V and Ib) based on the diversity of the fimA genes encoding the fimbrial subunits. In this study, fimA genotypic distribution was analyzed in P. gingivalis-infected cardiovascular specimens. METHODS A total of 112 heart valves and 80 atheromatous plaque specimens were collected from patients undergoing cardiovascular surgery, as well as 56 dental plaque specimens. Bacterial DNA was extracted from each, and polymerase chain reaction analysis was carried out with a P. gingivalis-specific set of primers. P. gingivalis-positive specimens were further analyzed to discriminate the fimA genotype using polymerase chain reaction with fimA type-specific primer sets. RESULTS P. gingivalis was detected in 10.4% of the cardiovascular specimens and 50.0% of the dental plaque samples. In the latter, type II was most frequently detected (35.7%), followed by types I (28.6%) and IV (21.4%), while types IV and II were detected with considerable frequencies of 45.0% and 30.0%, respectively, in the cardiovascular specimens. In contrast, the occurrence of type I was limited (5.0%) in the cardiovascular specimens. CONCLUSION These results suggest that specific fimA genotypic clones, which are reportedly associated with periodontitis, are also frequently harbored in cardiovascular specimens, indicating the possible involvement of type II and IV clones in the initiation and progression of cardiovascular diseases.

Upregulation of S100 calcium-binding protein A9 is required for induction of smooth muscle cell proliferation by a periodontal pathogen

We investigated the effect of a periodontal pathogen, Porphyromonas gingivalis, on human aortic smooth muscle cell (hAOSMC) proliferation as mechanisms of atherosclerosis. Cultured hAOSMCs exposed to the supernatant of plasma incubated with P. gingivalis showed a marked transformation from a contractile to proliferative phenotype, resulting in enhancement of cell growth. DNA microarray analysis revealed a P. gingivalis‐dependent upregulation of S100A9 in hAOSMCs. Small interference‐RNA for S100A9 dramatically attenuated the effect of P. gingivalis on transformation and proliferation of hAOSMCs. Our data suggested that upregulation of S100A9 mediated by P. gingivalis is an important event in the development of aortic intimal hyperplasia.

Porphyromonas gingivalis promotes invasion of oral squamous cell carcinoma through induction of proMMP9 and its activation

Recent epidemiological studies have revealed a significant association between periodontitis and oral squamous cell carcinoma (OSCC). Furthermore, matrix metalloproteinase 9 (MMP9) is implicated in the invasion and metastasis of tumour cells. We examined the involvement of Porphyromonas gingivalis, a periodontal pathogen, in OSCC invasion through induced expression of proMMP and its activation. proMMP9 was continuously secreted from carcinoma SAS cells, while P. gingivalis infection increased proenzyme expression and subsequently processed it to active MMP9 in culture supernatant, which enhanced cellular invasion. In contrast, Fusobacterium nucleatum, another periodontal organism, failed to demonstrate such activities. The effects of P. gingivalis were observed with highly invasive cells, but not with the low invasivetype. P. gingivalis also stimulated proteinase‐activated receptor 2 (PAR2) and enhanced proMMP9 expression, which promoted cellular invasion. P. gingivalis mutants deficient in gingipain proteases failed to activate MMP9. Infected SAS cells exhibited activation of ERK1/2, p38, and NF‐kB, and their inhibitors diminished both proMMP9‐overexpression and cellular invasion. Together, our results show that P. gingivalis activates the ERK1/2‐Ets1, p38/HSP27, and PAR2/NF‐kB pathways to induce proMMP9 expression, after which the proenzyme is activated by gingipains to promote cellular invasion of OSCC cell lines. These findings suggest a novel mechanism of progression and metastasis of OSCC associated with periodontitis.

Heterogenic virulence and related factors among clinical isolates of Porphyromonas gingivalis with type II fimbriae

BACKGROUND/AIMS Porphyromonas gingivalis is a periodontal pathogen whose fimbriae are classified into six genotypes (types I-V and Ib) based on the diversity of the fimA genes encoding the fimbrial subunits. Accumulated evidence suggests that P. gingivalis strains with type II fimbriae are more virulent as compared to those with other types. However, it is unknown if strong virulence is uniformly conserved among clones with type II fimbriae. In the present study, we compared infectious inflammatory changes in clinical isolates of P. gingivalis with type II fimbriae using a mouse abscess model to examine their pathogenic heterogeneity and heterogeneity-related factors. METHODS Suspensions of nine different clinical isolates with type II fimbriae were subcutaneously injected into female BALB/c mice and inflammatory parameters, such as serum sialic acid concentration, were compared. RESULTS Many of the type II fimbrial isolates caused severe inflammation in the mice, though some were less causative, as was the control strain ATCC 33277 (type I fimbria strain). These results showed that pathogenic heterogeneity exists among P. gingivalis clones with type II fimbriae. Further, the heterogeneity-related factors of P. gingivalis strains were analyzed and the pathogenic potentials showed positive relationships to gingipain activities and invasive efficiency but not to hydrophobicity or autoaggregation. In addition, invasive efficiency was related to the activities of gingipains that were extracellularly secreted. CONCLUSION These results suggest that pathogenic heterogeneity has relationships with the invasive and proteolytic activities of P. gingivalis clones with type II fimbriae.

Porphyromonas gingivalis invades human trophoblasts and inhibits proliferation by inducing G1 arrest and apoptosis

Porphyromonas gingivalis is an oral pathogen that is also associated with serious systemic conditions such as preterm delivery. Here we investigated the interaction between P. gingivalis and a cell line of extravillous trophoblasts (HTR‐8) derived from the human placenta. P. gingivalis internalized within HTR‐8 cells and inhibited proliferation through induction of arrest in the G1 phase of the cell cycle. G1 arrest was associated with decreased expression of cyclin D and of CDKs 2, 4 and 6. In addition, levels of CDK inhibitors p15, p16, p18 and p21 were increased following P. gingivalis infection. The amount of Rb was diminished by P. gingivalis, and transient overexpression of Rb, with concomitant upregulation of phospho‐Rb, relieved P. gingivalis‐induced G1 arrest. HTR‐8 cells halted in the G1 phase became apoptotic, and apoptosis was accompanied by an increase in the ratio of Bax/Bcl‐2 and increased activity of caspases 3, 7 and 9. HTR‐8 cells infected with P. gingivalis also exhibited a sustained activation of ERK1/2, and knock‐down of ERK1/2 activity with siRNA abrogated both G1 arrest and apoptosis. Thus, P. gingivalis can invade placental trophoblasts and induce G1 arrest and apoptosis through pathways involving ERK1/2 and its downstream effectors, properties that provide a mechanistic basis for pathogenicity in complications of pregnancy.