Pilus PilA of the naturally competent HACEK group pathogen Aggregatibacter actinomycetemcomitans stimulates human leukocytes and interacts with both DNA and proinflammatory cytokines.

Each HACEK group pathogen, which can cause infective endocarditis, expresses type IVa pili. The type IVa major pilin PilA plays a role in bacterial colonization, virulence, twitching motility, and the uptake of extracellular DNA. The type IV prepilin homolog PilA of the periodontal pathogen A. actinomycetemcomitans (AaPilA) is linked to DNA uptake and natural competence. Our aim was to investigate the virulence properties and immunogenic potential of AaPilA. Since Neisseria meningitidis PilE, which shares sequence similarity with AaPilA, participates in sequestering host cytokines, we examined the ability of AaPilA to interact with various cytokines. Moreover, we investigated the structural characteristics of AaPilA with molecular modeling. AaPilA was conserved among A. actinomycetemcomitans strains. One of the 18 different natural variants, PilAD7S, is present in naturally competent strains. This variant interacted with DNA and bound interleukin (IL)-8 and tumor necrosis factor (TNF)-α. Specific anti-AaPilA antibodies were present in A. actinomycetemcomitans-positive periodontitis patient sera, and the production of reactive oxygen species from human neutrophils was less effectively induced by the ΔpilA mutant than by the wild-type strains. However, AaPilA did not stimulate human macrophages to produce proinflammatory cytokines, nor was it cytotoxic. The results strengthen our earlier hypothesis that the DNA uptake machinery of A. actinomycetemcomitans is involved in the sequestration of inflammatory cytokines. Furthermore, AaPilA stimulates host immune cells, such as B cells and neutrophils, making it a potential virulence factor.

Schizophyllum commune ß-glucan: Effect on interleukin-10 expression induced by lipopolysaccharide from periodontopathic bacteria.

ß-glucans are potent immunomodulators, with effects on innate and adaptive immune responses via dectin-1 as the main receptor. In this study, we investigated the biological effect of ß-glucan from Schizophyllum commune, called Schizophyllan (SPG) on Interleukin-10 (IL-10) expression induced by a lipopolysaccharide (LPS) from Aggregatibacter actinomycetemcomitans in murine macrophages (J774.1). SPG and dectin-1 interaction up-regulates LPS-induced IL-10 expression. The regulative effect of SPG on IL-10 expression is dependent on prolongation of nuclear translocation activity of nuclear factor-kappa B (NF-κBα) pathway induced by LPS. We also found that LPS-induced phosphorylation of mitogen- and stress-activated protein kinase 1 (MSK1) and cAMP-responsive-element-binding protein (CREB), followed by up-regulation of IL-10, was stimulated by SPG priming via activation of the spleen tyrosine kinase (Syk). Our data indicate that SPG augments the anti-inflammatory response in murine macrophages which can be useful to create an intervention for periodontal disease treatment.

Extracellular RNAs in periodontopathogenic outer membrane vesicles promote TNF-α production in human macrophages and cross the blood-brain barrier in mice.

Among the main bacteria implicated in the pathology of periodontal disease, Aggregatibacter actinomycetemcomitans (Aa) is well known for causing loss of periodontal attachment and systemic disease. Recent studies have suggested that secreted extracellular RNAs (exRNAs) from several bacteria may be important in periodontitis, although their role is unclear. Emerging evidence indicates that exRNAs circulate in nanosized bilayered and membranous extracellular vesicles (EVs) known as outer membrane vesicles (OMVs) in gram-negative bacteria. In this study, we analyzed the small RNA expression profiles in activated human macrophage-like cells (U937) infected with OMVs from Aa and investigated whether these cells can harbor exRNAs of bacterial origin that have been loaded into the host RNA-induced silencing complex, thus regulating host target transcripts. Our results provide evidence for the cytoplasmic delivery and activity of microbial EV-derived small exRNAs in host gene regulation. The production of TNF-α was promoted by exRNAs via the TLR-8 and NF-κB signaling pathways. Numerous studies have linked periodontal disease to neuroinflammatory diseases but without elucidating specific mechanisms for the connection. We show here that intracardiac injection of Aa OMVs in mice showed successful delivery to the brain after crossing the blood-brain barrier, the exRNA cargos increasing expression of TNF-α in the mouse brain. The current study indicates that host gene regulation by microRNAs originating from OMVs of the periodontal pathogen Aa is a novel mechanism for host gene regulation and that the transfer of OMV exRNAs to the brain may cause neuroinflammatory diseases like Alzheimer's.-Han, E.-C., Choi, S.-Y., Lee, Y., Park, J.-W., Hong, S.-H., Lee, H.-J. Extracellular RNAs in periodontopathogenic outer membrane vesicles promote TNF-α production in human macrophages and cross the blood-brain barrier in mice.

Catechin-mediated restructuring of a bacterial toxin inhibits activity.

BACKGROUND: Catechins, polyphenols derived from tea leaves, have been shown to have antibacterial properties, through direct killing of bacteria as well as through inhibition of bacterial toxin activity. In particular, certain catechins have been shown to have bactericidal effects on the oral bacterium, Aggregatibacter actinomycetemcomitans, as well as the ability to inhibit a key virulence factor of this organism, leukotoxin (LtxA). The mechanism of catechin-mediated inhibition of LtxA has not been shown. METHODS: In this work, we studied the ability of six catechins to inhibit LtxA-mediated cytotoxicity in human white blood cells, using Trypan blue staining, and investigated the mechanism of action using a combination of techniques, including fluorescence and circular dichroism spectroscopy, confocal microscopy, and surface plasmon resonance. RESULTS: We found that all the catechins except (-)-catechin inhibited the activity of this protein, with the galloylated catechins having the strongest effect. Pre-incubation of the toxin with the catechins increased the inhibitory action, indicating that the catechins act on the protein, rather than the cell. The secondary structure of LtxA was dramatically altered in the presence of catechin, which resulted in an inhibition of toxin binding to cholesterol, an important initial step in the cytotoxic mechanism of the toxin. CONCLUSIONS: These results demonstrate that the catechins inhibit LtxA activity by altering its structure to prevent interaction with specific molecules present on the host cell surface. GENERAL SIGNIFICANCE: Galloylated catechins modify protein toxin structure, inhibiting the toxin from binding to the requisite molecules on the host cell surface.

Stimulatory effect of Aggregatibacter actinomycetemcomitans DNA on proinflammatory cytokine expression by human gingival fibroblasts.

OBJECTIVE: While different virulence factors have been reported of Aggregatibacter actinomycetemcomitans (Aa), there is little information about the stimulatory effect of its DNA. The main purpose of this study was to assess the inflammatory response of human gingival fibroblasts (HGFs) stimulated with A. actinomycetemcomitans DNA. DESIGN: Cytokine levels of IL-6, IL-1α and TNF-α were measured on the supernatant of HGFs activated with 10, 25, 50 and 100µg/ml DNA of Aa during 24h. Primary cultures of HGFs were infected with Aa and its DNA at different times and concentrations to compare its cytotoxic effect. Cell damage and adhesion of Aa to HGFs were evaluated under light microscopy and Scanning electron microscopy respectively. RESULTS: There was a statistical difference (p<0.05) in cytokine expression in HGFs activated by bacterial DNA with a dose dependent on IL-6 expression and a significantly elevated expression of IL-1α and TNF-α compared to Human DNA negative control. Substantial morphological alterations were observed after infection of A. actinomycetemcomitans in HGFs but not with bDNA exposure. Aggregatibacter actinomycetemcomitans showed a high rate of adhesion and cell damage to HGFs after 30min. CONCLUSIONS: Genomic DNA of A. actinomycetemcomitans could be a factor in the pathogenesis of periodontitis that might play a major role in the inflammatory response.

A novel intrinsically disordered outer membrane lipoprotein of Aggregatibacter actinomycetemcomitans binds various cytokines and plays a role in biofilm response to interleukin-1ß and interleukin-8.

Intrinsically disordered proteins (IDPs) do not have a well-defined and stable 3-dimensional fold. Some IDPs can function as either transient or permanent binders of other proteins and may interact with an array of ligands by adopting different conformations. A novel outer membrane lipoprotein, bacterial interleukin receptor I (BilRI) of the opportunistic oral pathogen Aggregatibacter actinomycetemcomitans binds a key gatekeeper proinflammatory cytokine interleukin (IL)-1ß. Because the amino acid sequence of the novel lipoprotein resembles that of fibrinogen binder A of Haemophilus ducreyi, BilRI could have the potential to bind other proteins, such as host matrix proteins. However, from the tested host matrix proteins, BilRI interacted with neither collagen nor fibrinogen. Instead, the recombinant non-lipidated BilRI, which was intrinsically disordered, bound various pro/anti-inflammatory cytokines, such as IL-8, tumor necrosis factor (TNF)-α, interferon (IFN)-γ and IL-10. Moreover, BilRI played a role in the in vitro sensing of IL-1ß and IL-8 because low concentrations of cytokines did not decrease the amount of extracellular DNA in the matrix of bilRI- mutant biofilm as they did in the matrix of wild-type biofilm when the biofilms were exposed to recombinant cytokines for 22 hours. BilRI played a role in the internalization of IL-1ß in the gingival model system but did not affect either IL-8 or IL-6 uptake. However, bilRI deletion did not entirely prevent IL-1ß internalization, and the binding of cytokines to BilRI was relatively weak. Thus, BilRI might sequester cytokines on the surface of A. actinomycetemcomitans to facilitate the internalization process in low local cytokine concentrations.

Regulation of matrix metalloproteinase secretion by green tea catechins in a three-dimensional co-culture model of macrophages and gingival fibroblasts.

OBJECTIVES: Elevated levels of matrix metalloproteinases (MMPs) have been associated with the active phases of tissue and bone destruction in periodontitis, an inflammatory disease characterized by a significant breakdown of tooth support. In the present study, we used a three-dimensional (3D) co-culture model of macrophages and gingival fibroblasts to investigate the ability of a green tea extract and its major constituent epigallocatechin-3-gallate (EGCG) to regulate the secretion of MMP-3, -8, and -9. METHODS: The 3D co-culture model was composed of gingival fibroblasts embedded in a type I collagen matrix overlaid with macrophages. Two arbitrary ratios were tested. The ratio composed of 1 macrophage to 10 fibroblasts was used to mimic a slightly inflamed periodontal site while the ratio composed of 10 macrophages to 1 fibroblast was used to mimic a severely inflamed periodontal site. The 3D co-culture model was pre-treated for 2h with either the green tea extract or EGCG. It was then stimulated with Aggregatibacter actinomycetemcomitans lipopolysaccharide (LPS). The model was also first stimulated with LPS for 2h and then incubated with the green tea extract or EGCG. The concentrations of secreted MMP-3, -8, and -9 were quantified by enzyme-linked immunoassays. RESULTS: When the 3D co-culture model was stimulated with A. actinomycetemcomitans LPS, the 10:1 ratio of macrophages to gingival fibroblasts was associated with a highest secretion of MMP-3 and -9 and, to a lesser extent, MMP-8, than the 1:10 ratio. Non-cytotoxic concentrations of the green tea extract or EGCG reduced the basal secretion levels of all three MMPs. A 2-h treatment with the green tea extract or EGCG prior to the stimulation with LPS resulted in a dose-dependent decrease in MMP secretion, with MMP-9 showing the most significant decrease. A decrease in MMP secretion was also observed when the green tea extract or EGCG was added following a 2-h stimulation with LPS. CONCLUSIONS: Our results suggested that green tea catechins, and more specifically EGCG, offer promising prospects for the development of a novel adjunctive treatment for periodontitis because of their ability to decrease the secretion of MMPs, which are important tissue-destructive enzymes produced by mucosal and immune cells.

Apoptotic Effects of the B Subunit of Bacterial Cytolethal Distending Toxin on the A549 Lung Cancer Cell Line.

Cytolethal distending toxin (CDT) is a secreted tripartite genotoxin produced by many pathogenic gram-negative bacteria. It is composed of three subunits, CdtA, CdtB and CdtC, and CdtB-associated deoxyribonuclease (DNase) activity is essential for the CDT toxicity. In the present study, to design a novel potentially antitumor drug against lung cancer, the possible mechanisms of cdtB anticancer properties were explored in the A549 human lung adenocarcinoma cell line. A recombinant plasmid pcDNA3.1/cdtB was constructed expressing CdtB of human periodontal bacterium Aggregatibacter actinomycetemcomitans and investigated for toxic properties in A549 cells and possible mechanisms. It was observed that plasmid pcDNA3.1/cdtB caused loss of cell viability, morphologic changes and induction of apoptosis. Furthermore, measurement of caspase activity indicated involvement of an intrinsic pathway of cell apoptosis. Consequently, the recombinant plasmid pcDNA3.1/cdtB may have potential as a new class of therapeutic agent for gene therapy of lung cancer.

Inhibitory effects of a novel cationic dodecapeptide [CL(14-25)] derived from cyanate lyase of rice on endotoxic activities of LPSs from Escherichia coli and periodontopathic Aggregatibacter actinomycetemcomitans.

OBJECTIVE: CL(14-25), a dodecapeptide of cyanate lyase from rice, is a novel cationic α-helical antimicrobial peptide. In this study, we examined inhibitory ability of CL(14-25) against endotoxic activities of lipopolysaccharides (LPSs) from Escherichia coli and periodontal pathogenic Aggregatibacter actinomycetemcomitans. METHODS: Endotoxin-neutralizing activity of CL(14-25) was evaluated by inhibition to induction of cytokine and nitric oxide in human aortic endothelial cells (HAECs) and RAW264 mouse macrophage cells, respectively. Protective effect of CL(14-25) was determined in mice against lethal toxicity of LPS. RESULTS: IL-6 in HAECs was induced by stimulation with LPS preparations of A. actinomycetemcomitans and E. coli tested in this study, and addition of CL(14-25) to the medium caused inhibition of their induction in a dose-dependent manner. CL(14-25) inhibited NO induction in RAW264 cells by a smooth type LPS of E. coli O55:B5 and an Rc type LPS of E. coli J5 as well as lipid A of E. coli R515 in a dose-dependent manner. Simultaneous injection of E. coli O55:B5 LPS and CL(14-25) in BALB/c mice resulted in prevention of lethal toxicity of the former. The results of a Limulus amebocyte lysate assay and surface plasmon resonance analysis of interaction between CL(14-25) and E. coli LPS or lipid A showed that CL(14-25) specifically binds to a lipid A moiety of LPS. CONCLUSION: The results of present study suggest that CL(14-25) has a potential to be used as a nutraceutical agent for periodontal therapy.

The Aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin Active Subunit CdtB Contains a Cholesterol Recognition Sequence Required for Toxin Binding and Subunit Internalization.

Induction of cell cycle arrest in lymphocytes following exposure to the Aggregatibacter actinomycetemcomitans cytolethal distending toxin (Cdt) is dependent upon the integrity of lipid membrane microdomains. Moreover, we have previously demonstrated that the association of Cdt with target cells involves the CdtC subunit which binds to cholesterol via a cholesterol recognition amino acid consensus sequence (CRAC site). In this study, we demonstrate that the active Cdt subunit, CdtB, also is capable of binding to large unilamellar vesicles (LUVs) containing cholesterol. Furthermore, CdtB binding to cholesterol involves a similar CRAC site as that demonstrated for CdtC. Mutation of the CRAC site reduces binding to model membranes as well as toxin binding and CdtB internalization in both Jurkat cells and human macrophages. A concomitant reduction in Cdt-induced toxicity was also noted, indicated by reduced cell cycle arrest and apoptosis in Jurkat cells and a reduction in the proinflammatory response in macrophages (interleukin 1ß [IL-1ß] and tumor necrosis factor alpha [TNF-α] release). Collectively, these observations indicate that membrane cholesterol serves as an essential ligand for both CdtC and CdtB and, further, that this binding is necessary for both internalization of CdtB and subsequent molecular events leading to intoxication of cells.

LFA-1-targeting Leukotoxin (LtxA; Leukothera®) causes lymphoma tumor regression in a humanized mouse model and requires caspase-8 and Fas to kill malignant lymphocytes.

Leukotoxin (LtxA) is a protein secreted from the oral bacterium Aggregatibacter actinomycetemcomitans. LtxA binds to the ß2 integrin lymphocyte-associated function antigen-1 (LFA-1) on human white blood cells (WBCs), resulting in cell death. LtxA is currently under investigation as a novel therapy (Leukothera(®)) for treating hematologic malignancies and autoimmune diseases. We show here that LtxA has potent in vivo anti-lymphoma activity in mice. LtxA caused complete regression of B-cell tumors and promoted long-term survival of mice. The mechanism of LtxA-mediated killing of malignant lymphocytes was further examined. We found that LtxA kills malignant lymphocytes by a novel mechanism requiring the death receptor Fas and caspase-8, but not Fas ligand (FasL) or caspase-9. We also determined that LFA-1 and Fas are closely associated on the cell surface and this proximity of LFA-1 and Fas could explain how signaling through an integrin can lead to cell death. In addition to LFA-1, this work reveals a second surface protein, Fas, that is critical for LtxA-mediated cell death. Knowledge of the mechanism of cell death induced by LtxA will facilitate the development and understanding of this potent experimental therapeutic agent.

Critical role of MKP-1 in lipopolysaccharide-induced osteoclast formation through CXCL1 and CXCL2.

UNLABELLED: Osteoclast (OC) progenitors (OCP) have been defined in the bone marrow (BM) as CD3(-)CD45R(B220)(-)GR1(-)CD11b(lo/)(-)CD115(+) (dOCP) and more recently in the peripheral blood (PB) as Lym(-)Ly6G(-)CD11b(+)Ly6C(+). These progenitors respond to stimuli, including LPS from periopathogenic Aggregatibacter actinomycetemcomitans, activating MAPK signaling, resulting in cytokine/chemokine-mediated osteoclastogenesis. Intracellular negative signaling pathways, including MAPK phosphatase-1 (MKP-1, gene Dusp1) deactivate MAPK pathways (p-p38 and p-JNK) and reduce inflammatory cytokines/chemokines. OBJECTIVE: To delineate the role of MKP-1 in chemokine-mediated OC formation using defined OC progenitor populations. Given its role in innate immune inflammatory signaling, we hypothesize that MKP-1 regulates LPS-induced OC formation from BM OCP through deregulated chemokines. METHODS: BM and PB from WT and Dusp1(-/-) female mice (8-12weeks) was obtained and sorted into defined progenitor populations. BM sorted dOCP were primed with MCSF and RANKL (48h), blocked with vehicle or chemokine blocking antibodies and stimulated with LPS (48-96h). TRAP assay and OC activity were measured for OC formation and activity following treatments. NanoString Array and qPCR were utilized for gene expression analysis. RESULTS: Dusp1(-/-) dOCPs formed more and larger osteoclasts from CD11b(hi) and dOCP compared to matched WT (P<0.05 each). PB-derived dOCP produced larger and more functional osteoclasts from Dusp1(-/-) mice compared to WT controls. NanoString array data revealed significant deregulation in chemokine expression from Dusp1(-/-) versus WT cells. qPCR validation of target genes revealed that Dusp1 deficient CD11b(+) populations display 1.5-3.5-fold greater expression of CXCL1 and 2-3-fold greater expression of CXCL2 compared to WT in CD11b(hi) and dOCP (P<0.05 each). Antibody blocking studies using anti-CXCL1 and CXCL2 antibodies blunted osteoclastogenesis in Dusp1(-/-) cells. CONCLUSION: MKP-1 negatively regulates chemokine-driven OC formation and subsequent bone resorption in response to LPS stimulation. Collectively, these data provide useful insight into mechanisms potentially leading to the development of therapeutic treatment of periodontal disease.

Opposite effect of supernatants from selected periopathogens and oral lactobacilli cultures on ATP levels in human gingival fibroblasts.

Studies were performed on the effects of supernatants obtained from bacterial cultures, including cultures of Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Prevotella intermedia and Lactobacillus acidophilus strains on ATP levels in human gingival fibroblasts (HGF-1) and on their viability. ATP levels were evaluated using luminescence test and cell viability was estimated using a fluorescence test. In control cultures mean levels of ATP in HGF-1 amounted to 4.90±0.32 mln RLU. Supernatants of P. gingivalis and A. actinomycetemcomitans cultures were found to significantly reduce ATP production in HGF-1 (mean levels of ATP amounted to 3.41±0.33 and 3.55±0.3 mln RLU respectively), which was not accompanied by an increased proportion of dead fibroblasts. Supernatants of P. intermedia induced no significant alterations in ATP level in HGF-1. In turn, supernatants of L. acidophilus H2O2 (+) and H2O2 (-) cultures significantly increased ATP levels in HGF-1 (the mean levels amounted to 5.94±0.31 mln RLU and 5.88±0.28 mln RLU respectively). The results indicate that extracellular products of P. gingivalis and A. actinomycetemcomitans most probably represent mitochondria-targeted peptides, which reduce synthesis of ATP in HGF-1. In turn, extracellular products of L. acidophilus seem to represent exopolysaccharides (EPS) with pro-oxidant activity, which stimulate synthesis of ATP in HGF-1.

The cytolethal distending toxin of Aggregatibacter actinomycetemcomitans inhibits macrophage phagocytosis and subverts cytokine production.

Aggregatibacter actinomycetemcomitans is an important periodontal pathogen that can participate in periodontitis and other non-oral infections. The cytolethal distending toxin (Cdt) is among the virulence factors produced by this bacterium. The Cdt is also secreted by several mucosa-associated Gram-negative pathogens and may play a role in perpetuating the infection by modulating the immune response. Although the toxin targets a wide range of eukaryotic cell types little is known about its activity on macrophages which play a key part in alerting the rest of the immune system to the presence of pathogens and their virulence factors. In view of this, we tested the hypothesis that the A. actinomycetemcomitans Cdt (AaCdt) disrupts macrophage function by inhibiting phagocytic activity as well as affecting the production of cytokines. Murine macrophages were co-cultured with either wild-type A. actinomycetemcomitans or a Cdt(-) mutant. Viable counts and qPCR showed that phagocytosis of the wild-type strain was significantly reduced relative to that of the Cdt(-) mutant. Addition of recombinant Aa(r)Cdt to co-cultures along with the Cdt(-) mutant diminished the phagocytic activity similar to that observed with the wild type strain. High concentrations of Aa(r)Cdt resulted in decreased phagocytosis of fluorescent bioparticles. Nitric oxide production was modulated by the presence of Cdt and the levels of IL-1ß, IL-12 and IL-10 were increased. Production of TNF-α did not differ in the co-culture assays but was increased by the presence of Aa(r)Cdt. These data suggest that the Cdt may modulate macrophage function in A. actinomycetemcomitans infected sites by impairing phagocytosis and modifying the pro-inflammatory/anti-inflammatory cytokine balance.

A. actinomycetemcomitans LPS enhances foam cell formation induced by LDL.

The objective of this study was to examine whether native low-density lipoprotein (LDL) induces foam cell formation by macrophages and to examine the effect of lipopolysaccharide (LPS) on native LDL-induced foam cell formation by macrophages in vitro. RAW 264.7 cells were cultured with LDL or high-density lipoprotein (HDL) in the presence of LPS derived from Aggregatibacter actinomycetemcomitans. Foam cell formation was determined by staining with Oil-red-O to visualize cytoplasmic lipid droplet accumulation. The expression of LDL-receptor and the degree of internalization of FITC-conjugated LDL in RAW 264.7 cells were examined by immunofluorescence microscopy. The images were digitally recorded and analyzed with Image J software. Statistical analysis was performed by JMP software. Foam cell formation was induced by the addition of native LDL in dose- and time-dependent manners, whereas HDL showed no effect. LPS enhanced the foam cell formation induced by native LDL. In addition, LPS stimulated the expression of LDL-receptor protein on RAW 264.7 cells and enhanced the internalization of LDL. The enhancement of foam cell formation induced by LPS and LDL was inhibited by the depolymerizing agent nocodazole and amiloride analog 5-(N-ethyl-N-isoprophyl) amiloride (EIPA). Our findings indicate that LPS plays an important role in foam cell formation by LDL-stimulated macrophages.

Aggregatibacter actinomycetemcomitans lipopolysaccharide regulates bone sialoprotein gene transcription.

Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) is believed to be associated with aggressive periodontitis characterized by a rapid bone loss. A. actinomycetemcomitans lipopolysaccharide (LPS) has a similar structure to Escherichia coli LPS, and they are Toll-like receptor 4 agonists. Bone sialoprotein (BSP) is an early marker of osteoblast differentiation. To investigate the effects of A. actinomycetemcomitans LPS on bone formation, we targeted BSP as a marker for osteogenic differentiation and bone formation. BSP mRNA levels were decreased by 0.1 µg/ml and increased by 0.01 µg/ml A. actinomycetemcomitans LPS at 6 h in osteoblast-like ROS17/2.8 cells. In transient transfection analyses, 0.1 µg/ml decreased and 0.01 µg/ml A. actinomycetemcomitans LPS increased luciferase activities of the construct (-116 to +60). Introduction of 2 bp mutations to the constructs showed that the effects of A. actinomycetemcomitans LPS were mediated by a cAMP response element (CRE), a FGF2 response element (FRE), and a homeodomain protein-binding site (HOX). Tyrosine kinase, ERK1/2, and PI3-kinase/Akt participated in the effects of both 0.1 and 0.01 µg/ml A. actinomycetemcomitans LPS. The results of gel shift showed that 0.1 µg/ml decreased while 0.01 µg/ml A. actinomycetemcomitans LPS increased CRE-, FRE-, and HOX-binding protein complexes formation at 6 h, and revealed that 0.01 µg/ml A. actinomycetemcomitans LPS induced BSP transcription through CREB1, JunD, Fra2, c-Fos, Runx2, Dlx5, and Smad1 targeting those response elements. These studies therefore indicated that 0.1 µg/ml suppressed and 0.01 µg/ml A. actinomycetemcomitans LPS increased BSP gene transcription mediated through CRE, FRE, and HOX elements in the rat BSP gene promoter.

Cytolethal distending toxin damages the oral epithelium of gingival explants.

UNLABELLED: The cytolethal distending toxin (Cdt), expressed by the periodontal pathogen Aggregatibacter actinomycetemcomitans, inhibits the proliferation of cultured epithelial cells by arresting the cell cycle. The gingival epithelium is an early line of defense against microbial assault. When damaged, bacteria collectively gain entry into underlying connective tissue where microbial products can affect infiltrating inflammatory cells, leading to the destruction of the attachment apparatus. Histological evaluation of rat and healthy human gingival tissue exposed ex vivo to the Cdt for 36 and 18 hours, respectively, revealed extensive detachment of the keratinized outer layer and distention of spinous and basal cells in the oral epithelium. Treated human tissue also exhibited disruption of rete pegs and dissolution of cell junctions. Cells in the connective tissue appeared unaffected. Primary gingival epithelial cells, but not gingival fibroblasts, isolated from the same healthy human tissue were cell-cycle-arrested when treated with the toxin. These findings provide new evidence that the Cdt severely damages the oral epithelium, ex vivo, by specifically targeting epithelial cells, in situ. The Cdt shows preferential targeting of the epithelium as opposed to connective tissue in animal and human gingival explant models. ABBREVIATIONS: cytolethal distending toxin (Cdt), connective tissue (CT), 4',6-diamidino-2-phenylindole (DAPI), human gingival epithelial cells (HGEC), human gingival explants (HGX), human gingival fibroblasts (HGF), junctional epithelium (JE), oral epithelium (OE), rete pegs (RP), sulcular epithelium (SE).

Periodontal pathogen Aggregatibacter actinomycetemcomitans LPS induces mitochondria-dependent-apoptosis in human placental trophoblasts.

OBJECTIVE: Increasing evidence suggests an association between periodontal disease and low birthweight (LBW); however the underlying molecular mechanisms are yet to be fully elucidated. In this study, we performed a microarray analysis to observe the human placental trophoblast-like BeWo cells response to lipopolysaccharide (LPS) from periodontopathogen Aggregatibacter actinomycetemcomitans (Aa), in order to investigate the molecular basis of mechanisms for periodontitis-associated LBW. In vivo pregnant rats were also used to confirm the in vitro results. STUDY DESIGN: The effects of Aa-LPS on cultured human placental trophoblast-like BeWo cells were studied using a DNA microarray, Ingenuity Pathway Analysis, real-time PCR and poly-caspase staining. The in vivo effects of Aa-LPS in pregnant rats were examined using TUNEL assays. RESULTS: In BeWo cells, Aa-LPS increased levels of cytochrome c, caspase 2, caspase 3, caspase 9 and BCL2-antagonist/killer 1 mRNA, decreased those of B-cell CLL/lymphoma 2, BCL2-like 1 and catalase mRNA and increased poly-caspase activity, all of which are consistent with activation of the mitochondria-dependent apoptotic pathway. TUNEL assays confirmed the increased incidence of apoptosis in placentas of Aa-LPS-treated rats (p < 0.001). CONCLUSION: Aa-LPS induces apoptosis in human trophoblasts via the mitochondria-dependent pathway, and this effect may contribute to the pathogenesis of periodontitis-associated LBW.

Role of the ATM-checkpoint kinase 2 pathway in CDT-mediated apoptosis of gingival epithelial cells.

The cytolethal distending toxin (CDT) of the oral pathogen Aggregatibacter actinomycetemcomitans induces cell cycle arrest and apoptosis in various cell types. Western analysis, pharmacological inhibition and siRNA silencing were performed in human immortalized gingival keratinocytes (HIGK) to dissect the functional role of the ataxia telangiectasia mutated (ATM) pathway in the signal transduction steps triggered by the CDT. Infection of HIGK was associated with a time-dependent induction of cytoplasmic histone-associated DNA fragmentation. However, in the absence of CDT, infected HIGK underwent reversible DNA strand breaks but not apoptosis, while caspase 3 activity, p21 levels, and HIGK viability were unaffected. Caspase 9 activity was attenuated in the CDT mutant-infected HIGK compared to wild-type infected cells. Pharmacological inhibition and siRNA-silencing of the ATM downstream effector, the protein kinase checkpoint kinase 2 (Chk2), significantly impacted CDT-mediated apoptosis. Together, these findings provide insight on the specificity of the ATM-Chk2 pathway in response to the CDT of A. actinomycetemcomitans in oral epithelial cells, which ultimately leads to apoptosis. We further propose the existence of an unidentified factor that is distinct from the CDT, and involved with a reversible DNA fragmentation that does not trigger terminal apoptosis in oral epithelial cells. This model potentially explains conflicting reports on the biological activity of the A. actinomycetemcomitans CDT.

Irsogladine maleate abolishes the increase in interleukin-8 levels caused by outer membrane protein 29 from Aggregatibacter (Actinobacillus) actinomycetemcomitans through the ERK pathway in human gingival epithelial cells.

BACKGROUND AND OBJECTIVE: Irsogladine maleate (IM) suppresses the increase in interleukin (IL)-8 production induced by outer membrane protein (OMP) 29 from Aggregatibacter (Actinobacillus) actinomycetemcomitans in cultures of human gingival epithelial cells (HGEC). However, how IM suppresses the OMP29-induced increase in IL-8 expression remains unknown. In this study, we focused on intracellular signaling pathways to elucidate the mechanism behind the suppression. MATERIAL AND METHODS: HGEC, which had been pretreated with inhibitors of intracellular signaling molecules, were exposed to OMP29 (1 microg/mL) with or without IM (1 microM). IL-8 expression at the mRNA and protein levels was examined by real-time polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Extracellular signal-regulated kinase (ERK) activity was measured with a p44/42 mitogen-activated protein kinase assay kit. RESULTS: An ERK inhibitor, PD98059, as well as IM, obviated the OMP29-induced increase in IL-8 levels in HGEC. A Jun kinase inhibitor, SP600125, and a nuclear factor kappaB inhibitor, PDTC, did not influence the OMP29-induced increase in IL-8 mRNA expression. The OMP29 stimulated phosphorylation of ERK in HGEC. Irsogladine maleate inhibited the phosphorylation. CONCLUSION: The suppression of the phosphorylation of ERK by IM in HGEC culminates in inhibition of the OMP29-induced increase in IL-8.