A polyphenolic cinnamon fraction exhibits anti-inflammatory properties in a monocyte/macrophage model.

Periodontal diseases are bacteria-induced inflammatory disorders that lead to the destruction of the tooth-supporting tissues. Active compounds endowed with a capacity to regulate the inflammatory response are regarded as potential therapeutic agents for the treatment of periodontal diseases. The aim of this study was to characterize the anti-inflammatory properties of a polyphenolic cinnamon fraction. Chromatographic and mass spectrometry analyses of the polyphenolic composition of the cinnamon fraction revealed that phenolic acids, flavonoids (flavonols, anthocyanins, flavan-3-ols), and procyanidins make up 9.22%, 0.72%, and 10.63% of the cinnamon fraction, respectively. We used a macrophage model stimulated with lipopolysaccharides (LPS) from either Aggregatibacter actinomycetemcomitans or Escherichia coli to show that the cinnamon fraction dose-dependently reduced IL-6, IL-8, and TNF-α secretion. Evidence was brought that this inhibition of cytokine secretion may result from the ability of the fraction to prevent LPS-induced NF-κB activation. We also showed that the cinnamon fraction reduces LPS binding to monocytes, which may contribute to its anti-inflammatory properties. Lastly, using a competitor assay, it was found that the cinnamon fraction may represent a natural PPAR-γ ligand. Within the limitations of this in vitro study, the cinnamon fraction was shown to exhibit a therapeutic potential for the treatment of periodontal diseases due to its anti-inflammatory properties.

Aggregatibacter actinomycetemcomitans Leukotoxin (LtxA; Leukothera®): Mechanisms of Action and Therapeutic Applications.

Aggregatibacter actinomycetemcomitans is an oral pathogen that produces the RTX toxin, leukotoxin (LtxA; Leukothera®). A. actinomycetemcomitans is strongly associated with the development of localized aggressive periodontitis. LtxA acts as a virulence factor for A. actinomycetemcomitans to subvert the host immune response by binding to the ß2 integrin lymphocyte function-associated antigen-1 (LFA-1; CD11a/CD18) on white blood cells (WBCs), causing cell death. In this paper, we reviewed the state of knowledge on LtxA interaction with WBCs and the subsequent mechanisms of induced cell death. Finally, we touched on the potential therapeutic applications of LtxA (trade name Leukothera®) toxin therapy for the treatment of hematological malignancies and immune-mediated diseases.

Aggregatibacter actinomycetemcomitans induces detachment and death of human gingival epithelial cells and fibroblasts via elastase release following leukotoxin-dependent neutrophil lysis.

Aggregatibacter actinomycetemcomitans is considered to be associated with periodontitis. Leukotoxin (LtxA), which destroys leukocytes in humans, is one of this bacterium's major virulence factors. Amounts of neutrophil elastase (NE), which is normally localized in the cytoplasm of neutrophils, are reportedly increased in the saliva of patients with periodontitis. However, the mechanism by which NE is released from human neutrophils and the role of NE in periodontitis is unclear. In the present study, it was hypothesized that LtxA induces NE release from human neutrophils, which subsequently causes the breakdown of periodontal tissues. LtxA-treatment did not induce significant cytotoxicity against human gingival epithelial cells (HGECs) or human gingival fibroblasts (HGFs). However, it did induce significant cytotoxicity against human neutrophils, leading to NE release. Furthermore, NE and the supernatant from LtxA-treated human neutrophils induced detachment and death of HGECs and HGFs, these effects being inhibited by administration of an NE inhibitor, sivelestat. The present results suggest that LtxA mediates human neutrophil lysis and induces the subsequent release of NE, which eventually results in detachment and death of HGECs and HGFs. Thus, LtxA-induced release of NE could cause breakdown of periodontal tissue and thereby exacerbate periodontitis.

Enhanced phagocytosis of Aggregatibacter actinomycetemcomitans cells by macrophages activated by a probiotic Lactobacillus strain.

The activation of phagocytosis is one important approach to clearing pathogenic cells in a host. This study evaluated the ability of probiotic lactobacilli to induce phagocytic activity as well as the clearance of a periodontal pathogen, Aggregatibacter actinomycetemcomitans. First, the activation of phagocytosis was found by using lyophilized dead cells. Probiotic Lactobacillus strains significantly enhanced the phagocytic activity of macrophage cells, indicating that the probiotic lactobacilli have a remarkable ability to stimulate the macrophages. Essentially, 3 Lactobacillus strains tested did not have any critical toxic effect on the murine macrophage, and Lactobacillus johnsonii NBRC 13952 showed the least cytotoxic effect on the RAW264.7 macrophages. The expression of classically activated macrophage markers, IL-1ß, and cluster of differentiation 80 increased by L. johnsonii NBRC 13952; however, there was no significant difference for IL-18. The highest phagocytic activity by macrophages was found in a condition in which the macrophage activated by L. johnsonii NBRC 13952 functions to kill the cells of A. actinomycetemcomitans. Correlating with the result, a high amount of hypodiploid DNA (SubG1) was detected from the macrophage cells stimulated by L. johnsonii NBRC 13952. Taken together, the results suggest that macrophages activated by the Lactobacillus strain can facilitate the phagocytosis of A. actinomycetemcomitans cells by linking with enhanced apoptotic activities. In conclusion, L. johnsonii NBRC 13952 has a certain role in activating the RAW264.7 macrophages, thereby counteracting the infection of A. actinomycetemcomitans.

Aggregatibacter (Actinobacillus) actimycetemcomitans leukotoxin and human periodontitis - A historic review with emphasis on JP2.

Aggregatibacter (Actinobacillus) actimycetemcomitans (Aa) is a gram-negative bacterium that colonizes the human oral cavity and is causative agent for localized aggressive (juvenile) periodontitis (AgP). In the middle of 1990s, a specific JP2 clone of belonging to the cluster of serotype b strains of Aa with highly leukotoxicity (leukotoxin, LtxA) able to kill human immune cells was isolated. JP2 clone of Aa was strongly associated with in particularly in rapidly progressing forms of aggressive periodontitis. The JP2 clone of Aa is transmitted through close contacts. Therefore, AgP patients need intense monitoring of their periodontal status as the risk for developing severely progressing periodontitis lesions are relatively high. Furthermore, timely periodontal treatment, including periodontal surgery supplemented by the use of antibiotics, is warranted. More importantly, periodontal attachment loss should be prevented by early detection of the JP2 clone of Aa by microbial diagnosis testing and/or preventive means.

Structure and mechanotransmission mechanism of the MacB ABC transporter superfamily.

MacB is an ABC transporter that collaborates with the MacA adaptor protein and TolC exit duct to drive efflux of antibiotics and enterotoxin STII out of the bacterial cell. Here we present the structure of ATP-bound MacB and reveal precise molecular details of its mechanism. The MacB transmembrane domain lacks a central cavity through which substrates could be passed, but instead conveys conformational changes from one side of the membrane to the other, a process we term mechanotransmission. Comparison of ATP-bound and nucleotide-free states reveals how reversible dimerization of the nucleotide binding domains drives opening and closing of the MacB periplasmic domains via concerted movements of the second transmembrane segment and major coupling helix. We propose that the assembled tripartite pump acts as a molecular bellows to propel substrates through the TolC exit duct, driven by MacB mechanotransmission. Homologs of MacB that do not form tripartite pumps, but share structural features underpinning mechanotransmission, include the LolCDE lipoprotein trafficking complex and FtsEX cell division signaling protein. The MacB architecture serves as the blueprint for understanding the structure and mechanism of an entire ABC transporter superfamily and the many diverse functions it supports.

Non-Surgical Therapy Reduces Presence of JP2 Clone in Localized Aggressive Periodontitis.

BACKGROUND: Previous studies have provided substantial evidence of the association of Aggregatibacter actinomycetemcomitans, and its highly leukotoxic JP2 genotype, with localized aggressive periodontitis (LAgP). The present study aims to evaluate presence of JP2 in individuals with LAgP after periodontal treatment. METHODS: Sixty African-American patients with LAgP, aged 5 to 25 years, were examined. At baseline, probing depth (PD), clinical attachment level (CAL), bleeding on probing, and plaque index were measured, and subgingival plaque was collected from LAgP diseased and healthy sites for each participant. Patients received whole-mouth ultrasonic debridement, scaling and root planing, and a 7-day prescription of amoxicillin and metronidazole. Participants were reevaluated and resampled and received regular maintenance therapy at 3, 6, and 12 months after treatment. Polymerase chain reaction was used to detect presence of the JP2 genotype before and after treatment. RESULTS: At baseline, the JP2 sequence was identified in 75% of LAgP diseased sites and in 56.67% of healthy sites. At 3, 6, and 12 months after treatment, the number of patients was 40, 31, and 31, respectively, and JP2 detection decreased to 17.5%, 6.45%, and 3.23%, respectively, in diseased sites (P <0.001) and to 2.5%, 3.23%, and 0%, respectively, in healthy sites (P <0.001). Clinical parameters of disease were also significantly reduced after therapy (P <0.001). Additionally, significant correlations were observed between JP2 presence and mean PD (P <0.002) and CAL (P <0.001), after therapy. CONCLUSION: Periodontal therapy was successful in reducing clinical parameters of LAgP and subgingival presence of JP2 in diseased and healthy sites.

Aggregatibacter actinomycetemcomitans regulates the expression of integrins and reduces cell adhesion via integrin α5 in human gingival epithelial cells.

Gingival epithelial cells form a physiological barrier against bacterial invasion. Excessive bacterial invasion destroys the attachment between the tooth surface and the epithelium, resulting in periodontitis. Integrins play a significant role in cell attachment; therefore, we hypothesized that bacterial infection might decrease the expressions of these integrins in gingival epithelial cells, resulting in reduced cell adhesion. Immortalized human gingival epithelial cells were co-cultured with Aggregatibacter actinomycetemcomitans Y4 (Aa Y4), and the gene expression levels of IL-8, proliferating cell nuclear antigen (PCNA), and integrins (α2, α3, α5, ß4, and ß6) were measured using quantitative reverse transcription polymerase chain reaction. Expression of PCNA and integrins, except integrin α5, was significantly downregulated, while expression of IL-8 and integrin α5 was significantly upregulated in the cells co-cultured with Aa Y4. The number of adherent cells significantly decreased when co-cultured with Aa Y4, as determined using cell adhesion assays. In the cells co-cultured with Aa Y4 and an integrin α5 neutralizing antibody, there was no effect on the expression of IL-8 and PCNA, while the expressions of integrins α2, α3, ß4, and ß6, and the number of adherent cells did not decrease. The number of invading bacteria in the cells was reduced in the presence of the antibody and increased in the presence of TLR2/4 inhibitor. Therefore, integrin α5 might be involved in Aa Y4 invasion into gingival epithelial cells, and the resulting signal transduction cascade reduces cell adhesion by decreasing the expression of integrins, while the TLR2/4 signaling cascade regulates IL-8 expression.

Some Gram-negative Lipoproteins Keep Their Surface Topology When Transplanted from One Species to Another and Deliver Foreign Polypeptides to the Bacterial Surface.

In Gram-negative bacteria, outer membrane-associated lipoproteins can either face the periplasm or protrude out of the bacterial surface. The mechanisms involved in lipoprotein transport through the outer membrane are not fully elucidated. Some lipoproteins reach the surface by using species-specific transport machinery. By contrast, a still poorly characterized group of lipoproteins appears to always cross the outer membrane, even when transplanted from one organism to another. To investigate such lipoproteins, we tested the expression and compartmentalization in E. coli of three surface-exposed lipoproteins, two from Neisseria meningitidis (Nm-fHbp and NHBA) and one from Aggregatibacter actinomycetemcomitans (Aa-fHbp). We found that all three lipoproteins were lipidated and compartmentalized in the E. coli outer membrane and in outer membrane vesicles. Furthermore, fluorescent antibody cell sorting analysis, proteolytic surface shaving, and confocal microscopy revealed that all three proteins were also exposed on the surface of the outer membrane. Removal or substitution of the first four amino acids following the lipidated cysteine residue and extensive deletions of the C-terminal regions in Nm-fHbp did not prevent the protein from reaching the surface of the outer membrane. Heterologous polypeptides, fused to the C termini of Nm-fHbp and NHBA, were efficiently transported to the E. coli cell surface and compartmentalized in outer membrane vesicles, demonstrating that these lipoproteins can be exploited in biotechnological applications requiring Gram-negative bacterial surface display of foreign polypeptides.

Real-time quantitative reverse transcription-PCR analysis of expression stability of Aggregatibacter actinomycetemcomitans fimbria-associated gene in response to photodynamic therapy.

BACKGROUND: Aggregatibacter actinomycetemcomitans is an etiological agent of both chronic and aggressive periodontitis. Dissemination of A. actinomycetemcomitans from the oral cavity and initiation of systemic infections has led to new approaches for treatment being needed. In this study, a series of experiments presented investigated the effect of methylene blue (MB)-mediated antimicrobial photodynamic therapy (aPDT) on cell viability and expression of fimbria-associated gene (rcpA) in A. actinomycetemcomitans. MATERIALS AND METHODS: To determine the dose-depended effects of aPDT, A. actinomycetemcomitans ATCC 33384 strain photosensitized with MB was irradiated with diode laser following bacterial viability measurements. Cell-surviving assay and expression ratio of rcpA were assessed by colony forming unit and real-time quantitative reverse transcription-PCR (qRT-PCR) assays, respectively. RESULTS: In the current study, MB-mediated aPDT using 100µg/mL showed significant reduction in A. actinomycetemcomitans growth when compared to the control (P<0.05). Sub-lethal dose of aPDT against A. actinomycetemcomitans was 25µg/mL MB at fluency of 93.75J/cm2. Sub-lethal dose of aPDT could lead to about four-fold suppression of expression of rcpA. CONCLUSION: High doses of MB-mediated aPDT could potentially exhibit antimicrobial activity, and the expression of rcpA as an important virulence factor of this strain is reduced in cells surviving aPDT with MB. So, aPDT can be a valuable tool for the treatment of A. actinomycetemcomitans infections.

The epithelial cell response to health and disease associated oral biofilm models.

BACKGROUND AND OBJECTIVE: Different bacteria differentially stimulate epithelial cells. Biofilm composition and viability are likely to influence the epithelial response. In vitro model systems are commonly used to investigate periodontitis-associated bacteria and their interactions with the host; therefore, understanding factors that influence biofilm-cell interactions is essential. The present study aimed to develop in vitro monospecies and multispecies biofilms and investigate the epithelial response to these biofilms. MATERIAL AND METHODS: Bacterial biofilms were cultured in vitro and then either live or methanol-fixed biofilms were co-cultured with epithelial cells. Changes in epithelial cell viability, gene expression and cytokine content of culture supernatants were evaluated. RESULTS: Bacterial viability was better preserved within mixed-species biofilm culture than within single-species biofilm culture. Both mixed- and single-species biofilms stimulated increased expression of mRNA for interleukin 8 (IL8), C-X-C motif chemokine ligand 3 (CXCL3), C-X-C motif chemokine ligand 1 (CXCL1), interleukin 1 (IL1), interleukin 6 (IL6), colony-stimulating factor 2 (CSF2) and tumour necrosis factor (TNF), and the response was greatest in response to mixed-species biofilms. Following co-culture, cytokines detected in the supernatants included IL-8, IL-6, granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor, with the greatest release of cytokines found following co-culture with methanol-fixed, mixed-species biofilms. CONCLUSIONS: These data show that epithelial cells generate a distinct cytokine gene- and protein-expression signature in response to live or fixed, single- or multispecies biofilms.

In vitro complement activation, adherence to red blood cells and induction of mononuclear cell cytokine production by four strains of Aggregatibacter actinomycetemcomitans with different fimbriation and expression of leukotoxin.

BACKGROUND AND OBJECTIVE: The periodontal pathogen Aggregatibacter actinomycetemcomitans has been proposed as pro-atherogenic, and complement-mediated adherence to red blood cells (RBCs) may facilitate its systemic spread. We investigated the ability of four strains of A. actinomycetemcomitans with differential expression of leukotoxin A (LtxA) and fimbriae to activate complement, adhere to RBCs and elicit cytokine responses by mononuclear cells (MNCs). MATERIAL AND METHODS: Aggregatibacter actinomycetemcomitans serotype b strains HK 921, HK 1651, HK 2092 and HK 2108 were fluorescence-labeled, incubated with human whole blood cells in the presence of autologous serum, and assessed for RBC adherence by flow cytometry and for capacity to induce cytokine production by cytometric bead array analysis. The levels of IgG to A. actinomycetemcomitans serotype b were quantified by ELISA, as was consumption of complement. RESULTS: The JP2 clone variants HK 1651 and, to a lesser extent, HK 2092, consumed complement efficiently, while HK 2108 (= strain Y4) consumed complement poorly. Nonetheless, the four tested strains adhered equally well to RBCs in the presence of autologous serum, without causing RBC lysis. The JP2 clone variant HK 2092, selectively lacking LtxA production, induced higher production of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6 and IL-10 by MNCs than did the other three strains, while the four strains induced similar production of IL-12p70. RBCs facilitated the HK 2092-induced production of TNF-α and IL-1ß, and IL-6 was enhanced by RBCs, and this facilitation could be counteracted by blockade of complement receptor 3 (CD11b/CD18). CONCLUSION: Our data suggest that the JP2 clone of A. actinomycetemcomitans, most closely resembled by the variant HK 1651, activates complement well, while strain Y4, represented by HK 2108, activates complement poorly. However, all strains of A. actinomycetemcomitans adhere to RBCs and, when capable of producing LtxA, prevent production of inflammatory cytokines by MNCs. This "immunologically silent" immune adherence may facilitate systemic spread and atherogenesis.

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.

Mobilization of TLR4 Into Lipid Rafts by Aggregatibacter Actinomycetemcomitans in Gingival Epithelial Cells.

BACKGROUND: An investigation of the mechanisms underlying the production of inflammatory cytokines through the stimulation of microorganisms on gingival epithelial cells may provide insights into the pathogenesis of the initiation of periodontitis. Lipid rafts, microdomains in the cell membrane, include a large number of receptors, and are centrally involved in signal transduction. We herein examined the involvement of lipid rafts in the expression of interleukin (IL-6) and IL-8 in gingival epithelial cells stimulated by periodontal pathogens. METHODS: OBA9, a human gingival cell line, was stimulated by Aggregatibacter actinomycetemcomitans or tumor necrosis factor (TNF)-α in the presence of methyl-ß-cyclodextrin (MßCD). RESULTS: A. actinomycetemcomitans or TNF-α increased IL-8 and IL-6 mRNA levels, and promoted the phosphorylation of ERK and p38 MAP kinase in OBA9. The pretreatment with MßCD abolished increases in IL-6 and IL-8 mRNA levels and the phosphorylation induced by A. actinomycetemcomitans, but did not suppress the response induced by TNF-α. The transfection of TLR4 inhibited A. actinomycetemcomitans-induced increases in IL-8 and IL-6 mRNA levels. Confocal microscopy revealed that MßCD inhibited the mobilization of TLR4 into lipid rafts. CONCLUSION: The mobilization of TLR4 into lipid rafts is involved in the expression of inflammatory cytokines and phosphorylation of MAP kinase in human gingival epithelial cells stimulated by A. actinomycetemcomitans.

A Commensal Bacterium Promotes Virulence of an Opportunistic Pathogen via Cross-Respiration.

UNLABELLED: Bacteria rarely inhabit infection sites alone, instead residing in diverse, multispecies communities. Despite this fact, bacterial pathogenesis studies primarily focus on monoculture infections, overlooking how community interactions influence the course of disease. In this study, we used global mutant fitness profiling (transposon sequencing [Tn-seq]) to determine the genetic requirements for the pathogenic bacterium Aggregatibacter actinomycetemcomitans to cause disease when coinfecting with the commensal bacterium Streptococcus gordonii Our results show that S. gordonii extensively alters A. actinomycetemcomitans requirements for virulence factors and biosynthetic pathways during infection. In addition, we discovered that the presence of S. gordonii enhances the bioavailability of oxygen during infection, allowing A. actinomycetemcomitans to shift from a primarily fermentative to a respiratory metabolism that enhances its growth yields and persistence. Mechanistically, respiratory metabolism enhances the fitness of A. actinomycetemcomitans in vivo by increasing ATP yields via central metabolism and creating a proton motive force. Our results reveal that, similar to cross-feeding, where one species provides another species with a nutrient, commensal bacteria can also provide electron acceptors that promote the respiratory growth and fitness of pathogens in vivo, an interaction that we term cross-respiration. IMPORTANCE: Commensal bacteria can enhance the virulence of pathogens in mixed-species infections. However, knowledge of the mechanisms underlying this clinically relevant phenomenon is lacking. To bridge this gap, we comprehensively determined the genes a pathogen needs to establish coinfection with a commensal. Our findings show that the metabolism of the pathogen is low-energy-yielding in monoinfection, but in coinfection, the commensal improves the fitness of the pathogen by increasing the bioavailability of oxygen, thereby shifting the pathogen toward a high-energy-yielding metabolism. Similar to cross-feeding, this interaction, which we term cross-respiration, illustrates that commensal bacteria can provide electron acceptors that enhance the virulence of pathogens during infection.

A Modified Glycosaminoglycan, GM-0111, Inhibits Molecular Signaling Involved in Periodontitis.

BACKGROUND: Periodontitis is characterized by microbial infection, inflammation, tissue breakdown, and accelerated loss of alveolar bone matrix. Treatment targeting these multiple stages of the disease provides ways to treat or prevent periodontitis. Certain glycosaminoglycans (GAGs) block multiple inflammatory mediators as well as suppress bacterial growth, suggesting that these GAGs may be exploited as a therapeutic for periodontitis. METHODS: We investigated the effects of a synthetic GAG, GM-0111, on various molecular events associated with periodontitis: growth of Porphyromonas gingivalis (P. gingivalis) and Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) pathogenic bacteria associated with periodontitis; activation of pro-inflammatory signaling through TLR2 and TLR4 in mouse macrophage RAW 264.7 cells and heterologously expressed HEK 293 cells; osteoclast formation and bone matrix resorption in cultured mouse pre-osteoclasts. RESULTS: (1) GM-0111 suppressed the growth of P. gingivalis and A. actinomycetemcomitans even at 1% (w/v) solution. The antibacterial effects of GM-0111 were stronger than hyaluronic acid (HA) or xylitol in P. gingivalis at all concentrations and comparable to xylitol in A. actinomycetemcomitans at ≥2% (w/v) solution. We also observed that GM-0111 suppressed biofilm formation of P. gingivalis and these effects were much stronger than HA. (2) GM-0111 inhibited TLR-mediated pro-inflammatory cellular signaling both in macrophage and HEK 293 cells with higher selectivity for TLR2 than TLR4 (IC50 of 1-10 ng/mL vs. > 100 µg/mL, respectively). (3) GM-0111 blocked RANKL-induced osteoclast formation (as low as 300 ng/mL) and bone matrix resorption. While GM-0111 showed high affinity binding to RANKL, it did not interfere with RANKL/RANK/NF-κB signaling, suggesting that GM-0111 inhibits osteoclast formation by a RANKL-RANK-independent mechanism. CONCLUSIONS: We report that GM-0111 inhibits multiple molecular events involved in periodontitis, spanning from the early pro-inflammatory TLR signaling, to pathways activated at the later stage component of bone loss.

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.

Alteration of Homeostasis in Pre-osteoclasts Induced by Aggregatibacter actinomycetemcomitans CDT.

The dysbiotic microbiota associated with aggressive periodontitis includes Aggregatibacter actinomycetemcomitans, the only oral species known to produce a cytolethal distending toxin (AaCDT). Give that CDT alters the cytokine profile in monocytic cells, we aimed to test the hypothesis that CDT plays a role in bone homeostasis by affecting the differentiation of precursor cells into osteoclasts. Recombinant AaCDT was added to murine bone marrow monocytes (BMMC) in the presence or absence of RANKL and the cell viability and cytokine profile of osteoclast precursor cells were determined. Multinucleated TRAP(+) cell numbers, and relative transcription of genes related to osteoclastogenesis were also evaluated. The addition of AaCDT did not lead to loss in cell viability but promoted an increase in the average number of TRAP(+) cells with 1-2 nuclei in the absence or presence of RANKL (Tukey, p < 0.05). This increase was also observed for TRAP(+) cells with ≥3nuclei, although this difference was not significant. Levels of TGF-ß, TNF-α, and IL-6, in the supernatant fraction of cells, were higher when in AaCDT exposed cells, whereas levels of IL-1ß and IL-10 were lower than controls under the same conditions. After interaction with AaCDT, transcription of the rank (encoding the receptor RANK), nfatc1 (transcription factor), and ctpK (encoding cathepsin K) genes was downregulated in pre-osteoclastic cells. The data indicated that despite the presence of RANKL and M-CSF, AaCDT may inhibit osteoclast differentiation by altering cytokine profiles and repressing transcription of genes involved in osteoclastogenesis. Therefore, the CDT may impair host defense mechanisms in periodontitis.

Aggregatibacter actinomycetemcomitans outer membrane protein 29 (Omp29) induces TGF-ß-regulated apoptosis signal in human gingival epithelial cells via fibronectin/integrinß1/FAK cascade.

Gingival junctional epithelial cell apoptosis caused by periodontopathic bacteria exacerbates periodontitis. This pathological apoptosis is involved in the activation of transforming growth factor ß (TGF-ß). However, the molecular mechanisms by which microbes induce the activation of TGF-ß remain unclear. We previously reported that Aggregatibacter actinomycetemcomitans (Aa) activated TGF-ß receptor (TGF-ßR)/smad2 signalling to induce epithelial cell apoptosis, even though Aa cannot bind to TGF-ßR. Additionally, outer membrane protein 29 kDa (Omp29), a member of the Aa Omps family, can induce actin rearrangements via focal adhesion kinase (FAK) signalling, which also plays a role in the activation of TGF-ß by cooperating with integrin. Accordingly, we hypothesized that Omp29-induced actin rearrangements via FAK activity would enhance the activation of TGF-ß, leading to gingival epithelial cell apoptosis in vitro. By using human gingival epithelial cell line OBA9, we found that Omp29 activated TGF-ßR/smad2 signalling and decreased active TGF-ß protein levels in the extracellular matrix (ECM) of cell culture, suggesting the transactivation of TGF-ßR. Inhibition of actin rearrangements by cytochalasin D or blebbistatin and knockdown of FAK or integrinß1 expression by siRNA transfection attenuated TGF-ßR/smad2 signalling activity and reduction of TGF-ß levels in the ECM caused by Omp29. Furthermore, Omp29 bound to fibronectin (Fn) to induce its aggregation on integrinß1, which is associated with TGF-ß signalling activity. All the chemical inhibitors and siRNAs tested blocked Omp29-induced OBA9 cells apoptosis. These results suggest that Omp29 binds to Fn in order to facilitate Fn/integrinß1/FAK signalling-dependent TGF-ß release from the ECM, thereby inducing gingival epithelial cell apoptosis via TGF-ßR/smad2 pathway.

Profound Effects of Aggregatibacter actinomycetemcomitans Leukotoxin Mutation on Adherence Properties Are Clarified in in vitro Experiments.

Leukotoxin (Ltx) is a prominent virulence factor produced by Aggregatibacter actinomycetemcomitans, an oral microorganism highly associated with aggressive periodontitis. Ltx compromises host responsiveness by altering the viability of neutrophils, lymphocytes, and macrophages. Previously, we developed a Rhesus (Rh) monkey colonization model designed to determine the effect of virulence gene mutations on colonization of A. actinomycetemcomitans. Unexpectedly, an A. actinomycetemcomitans leukotoxin (ltxA) mutant (RhAa-VS2) failed to colonize in the Rh model. No previous literature suggested that Ltx was associated with A. actinomycetemcomitans binding to tooth surfaces. These results led us to explore the broad effects of the ltxA mutation in vitro. Results indicated that LtxA activity was completely abolished in RhAa-VS2 strain, while complementation significantly (P<0.0001) restored leukotoxicity compared to RhAa-VS2 strain. RT-PCR analysis of ltx gene expression ruled out polar effects. Furthermore, binding of RhAa-VS2 to salivary-coated hydroxyapatite (SHA) was significantly decreased (P<0.0001) compared to wild type RhAa3 strain. Real time RT-PCR analysis of the genes related to SHA binding in RhAa-VS2 showed that genes related to binding were downregulated [rcpA (P = 0.018), rcpB (P = 0.02), tadA (P = 0.002)] as compared to wild type RhAa3. RhAa-VS2 also exhibited decreased biofilm depth (P = 0.008) and exo-polysaccharide production (P<0.0001). Buccal epithelial cell (BEC) binding of RhAa-VS2 was unaffected. Complementation with ltxA restored binding to SHA (P<0.002) but had no effect on biofilm formation when compared to RhAa3. In conclusion, mutation of ltxA diminished hard tissue binding in vitro, which helps explain the previous in vivo failure of a ltxA knockout to colonize the Rh oral cavity. These results suggest that; 1) one specific gene knockout (in this case ltxA) could affect other seemingly unrelated genes (such as rcpA, rcpB tadA etc), and 2) some caution should be used when interpreting the effect attributed to targeted gene mutations when seen in a competitive in vivo environment.