Muneaki Tamura

Identification of a Treponema denticola OppA Homologue That Binds Host Proteins Present in the Subgingival Environment

ABSTRACT Proteins secreted or exported by Treponema denticolahave been implicated as mediators of specific interactions between the spirochete and subgingival tissues in periodontal diseases. However, limited information is available on the ability of this peptidolytic organism to bind or transport soluble peptides present in the subgingival environment. A prominent 70-kDa protein was isolated from surface extracts of T. denticola ATCC 35405. A clone expressing a portion of the protein was identified in anEscherichia coli expression library of T. denticola DNA. DNA sequence analysis showed that the cloned gene encoded a peptide homologous to OppA, the solute binding protein of an ATP-binding cassette-type peptide transporter involved in peptide uptake and environmental signaling in a wide range of bacteria. Genes encoding OppB, -C, -D, and -F were identified directly downstream ofoppA in T. denticola. OppA was present in representative strains of T. denticola and inTreponema vincentii but was not detected in Treponema pectinovorum or Treponema socranskii. Immunogold electron microscopy suggested that OppA was accessible to proteins at the surface of the spirochete. Native OppA bound soluble plasminogen and fibronectin but did not bind to immobilized substrates or epithelial cells. A T. denticola oppA mutant bound reduced amounts of soluble plasminogen, and plasminogen binding to the parent strain was inhibited by the lysine analog ɛ-aminocaproic acid. Binding of soluble host proteins by OppA may be important both for spirochete-host interactions in the subgingival environment and for uptake of peptide nutrients.

Reactivation of latent HIV-1 by a wide variety of butyric acid-producing bacteria

Latently infected cells harbor human immunodeficiency virus type 1 (HIV-1) proviral DNA copies integrated in heterochromatin, allowing persistence of transcriptionally silent proviruses. It is widely accepted that hypoacetylation of histone proteins by histone deacetylases (HDACs) is involved in maintaining the HIV-1 latency by repressing viral transcription. HIV-1 replication can be induced from latently infected cells by environmental factors, such as inflammation and co-infection with other microbes. It is known that a bacterial metabolite butyric acid inhibits catalytic action of HDAC and induces transcription of silenced genes including HIV-1 provirus. There are a number of such bacteria in gut, vaginal, and oral cavities that produce butyric acid during their anaerobic glycolysis. Since these organs are known to be the major site of HIV-1 transmission and its replication, we explored a possibility that explosive viral replication in these organs could be ascribable to butyric acid produced from anaerobic resident bacteria. In this study, we demonstrate that the culture supernatant of various bacteria producing butyric acid could greatly reactivate the latently-infected HIV-1. These bacteria include Fusobacterium nucleatum (commonly present in oral cavity, and gut), Clostridium cochlearium, Eubacterium multiforme (gut), and Anaerococcus tetradius (vagina). We also clarified that butyric acid in these culture supernatants could induce histone acetylation and HIV-1 replication by inhibiting HDAC. Our observations indicate that butyric acid-producing bacteria could be involved in AIDS progression by reactivating the latent HIV provirus and, subsequently, by eliminating such bacterial infection may contribute to the prevention of the AIDS development and transmission.

Cell-substrate impedance analysis of epithelial cell shape and micromotion upon challenge with bacterial proteins that perturb extracellular matrix and cytoskeleton

Abstract Research into the nature of host cell–microbial cross-talk needs more sensitive methods to analyze real time cellular reactions to infection or to toxic factors from infecting agents. Here, we introduce novel analytical applications of electrical cell–substrate impedance sensing (ECIS) to quantify changes in cell morphology and micromotion in response to bacterial challenge. Epithelial monolayers were challenged with an outer membrane (OM) extract of Treponema denticola, its purified 95 kDa chymotrypsin, and major surface protein (Msp). Impedance changes were analyzed and discriminated through model calculation as either junctional resistance (Rb) or average cell–substrate separation (h). Fluctuations in impedance and the normalized variance ratio were used to quantify micromotion. The OM and the 95 kDa protease caused a rapid drop in impedance. Msp had no effect on Rb and h, but contributed to the reduction in micromotion. These new analytical applications of ECIS can be used to study toxigenic activities of any pathogen that affects cell junctions, cytoskeletal integrity, cell shape, or contact with extracellular matrix.

Zinc and copper play a role in coaggregation inhibiting action of Porphyromonas gingivalis.

BACKGROUND/AIM We investigated the mechanisms of adherence of salivary and serum proteins, which mimic gingival crevicular fluid (GCF), to Porphyromonas gingivalis, and the effects of these adhered proteins on coaggregation and hemagglutination properties. METHODS The amounts of salivary and serum proteins adhering to P. gingivalis were determined using (3)H-labeled and non-labeled proteins. The coaggregation between P. gingivalis and Streptococcus oralis or Streptococcus gordonii was observed. Hemagglutination was evaluated using sheep erythrocytes. Proteins that interacted with zinc or copper in saliva and serum and on P. gingivalis were examined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. RESULTS The amount of salivary or serum proteins that adhered to the surface of P. gingivalis strains was increased by cations, especially zinc and copper ions. The pretreatment of bacterial cells with salivary or serum proteins before the assay inhibited coaggregation with gram-positive bacteria and hemagglutination. These phenomena were enhanced by the presence of zinc or copper ions during the pretreatment of P. gingivalis with proteins. We detected protein bands that were related to these cations in saliva and serum and on P. gingivalis. CONCLUSIONS Our findings suggest that zinc and copper ions markedly enhanced the adhesion and accumulation of salivary and serum proteins on cells of P. gingivalis and inhibited the coaggregation and hemagglutination of P. gingivalis. These cations might be useful for limiting the settlement of P. gingivalis in the gingival sulcus with the goal of preventing periodontal disease.

Middle-aged rats orally supplemented with gel-encapsulated catechin favorably increases blood cytosolic NADPH levels

Green tea catechins are primarily known to function as free radical scavengers and have several beneficial uses. Orally supplemented catechin (OSC) was previously shown to increase mitochondrial heme and catalase levels in rat heart blood, however, its effect in the cytosol has not been elucidated. Here, we determined the effects of OSC in the rat heart blood cytosol. We used middle-aged (40 week-old) and young (4 week-old) rats throughout the study. We isolated blood cytosol, verified its purity, and determined heme, hydrogen peroxide (H2O2) levels, catalase (CAT) activities, gp91(phox) amounts, NADP and NAD pools, sirtuin 1 (SIRT1) and glutathione reductase (GR) activities, and free fatty acids (FFA). We established that OSC is associated with decreased heme-dependent H2O2 amounts while increasing heme-independent CAT activity. Moreover, we found that OSC-related decrease in NAD(+) amounts among middle-aged rats is associated to increased NADPH levels and SIRT1 activity. In contrast, we associated OSC-related decrease in NAD(+) amounts among young rats to decreased NADPH levels and increased SIRT1 activity. This highlights a major difference between catechin-treated middle-aged and young rats. Furthermore, we observed that cytosolic FFA and GR levels were significantly increased only among OSC-treated middle-aged rats which we hypothesize are related to increased NADPH levels. This insinuates that OSC treatment allows higher catechin amounts to enter the bloodstream of middle-aged rats. We propose that this would favorably increase NADPH amounts and lead to the simultaneous decrease in NADPH-related pro-oxidant activity and increase in NADPH-related biomolecules and anti-oxidant activities.

Bactericidal effect of hydroxyl radicals generated from a low concentration hydrogen peroxide with ultrasound in endodontic treatment

One approach to enhance the disinfection of root canals in endodontic treatment is ultrasonic irrigation with sodium hypochlorite. Reactive oxygen species, such as hydroxyl radical, are generated by biological defense systems to kill invading bacteria. Ultrasonic irrigation with hydrogen peroxide may be a promising option to increase hydroxyl radical generation. We examined the bactericidal effects of hydroxyl radical generated from low concentration hydrogen peroxide with ultrasound in vitro. An ultrasonic tip was submerged in 0.5 or 1.0 M hydrogen peroxide in a microfuge tube. hydrogen peroxide was irradiated with the ultrasound, the tip of which was maintained centered in the tube to mimic ultrasonic irrigation. Hydroxyl radical generation was assessed by electron spin resonance spectroscopy. Subsequently, Enterococcus faecalis suspension in hydrogen peroxide was prepared and irradiated as described above. Bactericidal effects were assessed by viable counting. Electron spin resonance measurements showed that hydroxyl radical generation increased significantly in a time- and dose-dependent manner (two-way analysis of variance and Tukey’s test, p<0.05). Moreover, the bactericidal effects of hydrogen peroxide against Enterococcus faecalis were enhanced by ultrasonic irradiation in a time- and dose-dependent manner. These results suggest that ultrasonic irrigation in the presence of low concentration hydrogen peroxide can serve as a disinfection strategy in endodontic treatment.

Neuraminidase-producing oral mitis group streptococci potentially contribute to influenza viral infection and reduction in antiviral efficacy of zanamivir

Influenza is a serious respiratory disease among immunocompromised individuals, such as the elderly, and its prevention is an urgent social issue. Influenza viruses rely on neuraminidase (NA) activity to release progeny viruses from infected cells and spreading the infection. NA is, therefore, an important target of anti-influenza drugs. A causal relationship between bacteria and influenza virus infection has not yet been established, however, a positive correlation between them has been reported. Thus, in this study, we examined the biological effects of oral mitis group streptococci, which are predominant constituents of human oral florae, on the release of influenza viruses. Among them, Streptococcusoralis ATCC 10557 and Streptococcus mitis ATCC 6249 were found to exhibit NA activity and their culture supernatants promoted the release of influenza virus and cell-to-cell spread of the infection. In addition, culture supernatants of these NA-producing oral bacteria increased viral M1 protein expression levels and cellular ERK activation. These effects were not observed with culture supernatants of Streptococcus sanguinis ATCC 10556 which lacks the ability to produce NA. Although the NA inhibitor zanamivir suppressed the release of progeny viruses from the infected cells, the viral release was restored upon the addition of culture supernatants of NA-producing S. oralis ATCC 10557 or S. mitis ATCC 6249. These findings suggest that an increase in the number of NA-producing oral bacteria could elevate the risk of and exacerbate the influenza infection, hampering the efficacy of viral NA inhibitor drugs.

Orally supplemented catechin increases heme amounts and catalase activities in rat heart blood mitochondria: A comparison between middle-aged and young rats

Orally-administered catechin has long been known to have several beneficial effects on the mammalian host, however, the effects of orally supplemented catechin on the host through gingival tissues have not yet been established. Here, we elucidated the effects of orally supplemented catechin in the rat heart blood mitochondria. We used middle-aged (40 week-old) and young (4 week-old) rats throughout the study. We indirectly verified blood serum catechin levels by measuring O-methyl catechin derivatives using HPLC. Interestingly, we observed higher blood serum O-methyl levels in middle-aged rats as compared to young rats. Subsequently, we isolated blood mitochondria, verified its purity, and measured heme, hydrogen peroxide, and catalase (CAT) levels. We found that catechin induces an increase in blood mitochondrial heme amounts and is associated with an increase in blood mitochondrial CAT activity which is surprisingly higher in middle-aged rats as compared to young rats. This would imply that orally supplemented catechin induces heme increase that preferentially favours CAT activity and is more beneficial to the middle-aged rats.

Butyric Acid Effects in the Development of Periodontitis and Systemic Diseases

Abstract The common metabolic by-product of periodontopathic bacteria, butyric acid, not only causes many varying different effects on mammalian cells, but also results in the induction of microbial pathogenicity and reactivation of latent virus. A high concentration of butyric acid induces cytotoxicity and apoptosis among T cells, B cells and inflamed fibroblasts. Likewise, it contributes to the destruction of gingival tissue and modulation of local immunity at gingival sites. Moreover, butyric acid promoted cancer cell migration, suggesting that the progression of periodontal diseases may promote the progression of oral cancer. Epigenetic regulation is involved in the establishment and maintenance of HIV-1 latency and HIV-1 reactivation by butyric acid. It may be that butyric acid could contribute to the destruction of gingival tissues, alter immunomodulation and play an important role in certain systemic diseases. This review focuses on the effects of commensal bacteria at gingival sites ; in particular, (1) butyric acid on the cells of host tissue, (2) microbial cells, and (3) cancer cells in the onset of systemic infection.

Ab initio modeling approach towards establishing the structure and docking orientation of the Porphyromonas gingivalis FimA.

Porphyromonas gingivalis FimA is a major aetiological agent in periodontal disease development, however, its structure has never been determined. Here, we established the mature P. gingivalis FimA ab initio model of all six FimA variants. We determined the conserved amino acid sequences of each FimA variant and generated mature FimA models. Subsequently, we validated their quality, protein empirical distribution, and radius of gyration. Similarly, structural comparison and topological orientation were elucidated, and the probable protein-protein docking was investigated. We found that the putative mature FimA model is β-sheet-rich and, likewise, we observed that each mature FimA model has varying levels of structural differences which can be topologically subdivided into the upper, middle, and lower FimA sections. Moreover, we found that the FimA epithelial cell-binding domain (EBD) is structurally conserved within the middle FimA section of all variants and FimA-FimA docking suggests that the FimA EBDs are oriented in opposite and alternating directions of each other.