Casey Chen

The Cytolethal Distending Toxin Induces Receptor Activator of NF-κB Ligand Expression in Human Gingival Fibroblasts and Periodontal Ligament Cells

ABSTRACT Actinobacillus actinomycetemcomitans is associated with localized aggressive periodontitis, a disease characterized by rapid loss of the alveolar bone surrounding the teeth. Receptor activator of NF-κB Ligand (RANKL) and osteoprotegerin (OPG) are two molecules that regulate osteoclast formation and bone resorption. RANKL induces osteoclast differentiation and activation, whereas OPG blocks this process by acting as a decoy receptor for RANKL. The purpose of this study was to investigate the effect of A. actinomycetemcomitans on the expression of RANKL and OPG in human gingival fibroblasts and periodontal ligament cells. RANKL mRNA expression was induced in both cell types challenged by A. actinomycetemcomitans extract, whereas OPG mRNA expression remained unaffected. Cell surface RANKL protein was also induced by A. actinomycetemcomitans, whereas there was no change in OPG protein secretion. A cytolethal distending toxin (Cdt) gene-knockout strain of A. actinomycetemcomitans did not induce RANKL expression, in contrast to its wild-type strain. Purified Cdt from Haemophilus ducreyi alone, or in combination with extract from the A. actinomycetemcomitans cdt mutant strain, induced RANKL expression. Pretreatment of A. actinomycetemcomitans wild-type extract with Cdt antiserum abolished RANKL expression. In conclusion, A. actinomycetemcomitans induces RANKL expression in periodontal connective tissue cells. Cdt is crucial for this induction and may therefore be involved in the pathological bone resorption during the process of localized aggressive periodontitis.

Proinflammatory effect in whole blood by free soluble bacterial components released from planktonic and biofilm cells

BackgroundAggregatibacter actinomycetemcomitans is an oral bacterium associated with aggressive forms of periodontitis. Increasing evidence points to a link between periodontitis and cardiovascular diseases, however, the underlying mechanisms are poorly understood. This study investigated the pathogenic potential of free-soluble surface material, released from live planktonic and biofilm A. actinomycetemcomitans cells.ResultsBy employing an ex vivo insert model (filter pore size 20 nm) we demonstrated that the A. actinomycetemcomitans strain D7S and its derivatives, in both planktonic and in biofilm life-form, released free-soluble surface material independent of outer membrane vesicles. This material clearly enhanced the production of several proinflammatory cytokines (IL-1β, TNF-α, IL-6, IL-8, MIP-1β) in human whole blood, as evidenced by using a cytokine antibody array and dissociation-enhanced-lanthanide-fluorescent-immunoassay. In agreement with this, quantitative real-time PCR indicated a concomitant increase in transcription of each of these cytokine genes. Experiments in which the LPS activity was blocked with polymyxin B showed that the stimulatory effect was only partly LPS-dependent, suggesting the involvement of additional free-soluble factors. Consistent with this, MALDI-TOF-MS and immunoblotting revealed release of GroEL-like protein in free-soluble form. Conversely, the immunomodulatory toxins, cytolethal distending toxin and leukotoxin, and peptidoglycan-associated lipoprotein, appeared to be less important, as evidenced by studying strain D7S cdt/ltx double, and pal single mutants. In addition to A. actinomycetemcomitans a non-oral species, Escherichia coli strain IHE3034, tested in the same ex vivo model also released free-soluble surface material with proinflammatory activity.ConclusionA. actinomycetemcomitans, grown in biofilm and planktonic form, releases free-soluble surface material independent of outer membrane vesicles, which induces proinflammatory responses in human whole blood. Our findings therefore suggest that release of surface components from live bacterial cells could constitute a mechanism for systemic stimulation and be of particular importance in chronic localized infections, such as periodontitis.

Cell cycle arrest of human gingival fibroblasts and periodontal ligament cells by Actinobacillus actinomycetemcomitans: involvement of the cytolethal distending toxin†

The cytolethal distending toxin (Cdt) is produced by several Gram‐negative bacterial species and causes growth arrest and morphological alterations in mammalian cells. Actinobacillus actinomycetemcomitans, which is involved in the pathogenesis of localized aggressive periodontitis, also produces a Cdt that affects periodontal connective tissue cells. The aim of this study was to investigate in which phase of the cell cycle these cells are arrested and enlarged when challenged with A. actinomycetemcomitans, and to evaluate the involvement of its Cdt. Human gingival fibroblasts and periodontal ligament cells were challenged with A. actinomycetemcomitans extract, or with purified Cdt, and cell cycle analysis was performed by propidium iodide staining and flow cytometry. Cells exposed to an A. actinomycetemcomitans wild‐type strain, or to purified Cdt, were arrested in both G1 and G2/M phases, and appeared enlarged compared to the corresponding controls. The cellular enlargement occurred in both G1 and G2/M arrested cells. In contrast, cells exposed to an A. actinomycetemcomitanscdt‐knockout mutant strain showed cell cycle phase distribution and size similar to the controls. In conclusion, A. actinomycetemcomitans causes a combined G1 and G2/M growth arrest and enlargement in periodontal connective tissue cells, which is attributed to its Cdt.

Genome Sequence of Aggregatibacter actinomycetemcomitans Serotype c Strain D11S-1

Aggregatibacter actinomycetemcomitans is a major etiological agent of periodontitis. Here we report the complete genome sequence of serotype c strain D11S-1, which was recovered from the subgingival plaque of a patient diagnosed with generalized aggressive periodontitis.

Genome Sequence of Naturally Competent Aggregatibacter actinomycetemcomitans Serotype a Strain D7S-1

The major clonal lineages of the Gram-negative periodontal pathogen Aggregatibacter actinomycetemcomitans include serotype a, b, and c strains. Here, we report the draft genome sequence of a naturally competent serotype a strain, D7S-1, isolated from a patient with aggressive periodontitis.

Development of an animal model for Aggregatibacter actinomycetemcomitans biofilm-mediated oral osteolytic infection: a preliminary study.

BACKGROUND Biofilm-induced inflammatory osteolytic oral infections, such as periodontitis and peri-implantitis, have complex etiology and pathogenesis. A significant obstacle to research has been the lack of appropriate animal models where the inflammatory response to biofilms can be investigated. The aim of this study is to develop a novel animal model to study the host response to Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans)-biofilm colonizing titanium implants. METHODS Titanium implants were inoculated in vitro with A. actinomycetemcomitans, establishing a biofilm for 1 to 3 days. Biofilm-inoculated and control implants were transmucosally placed into rat hard palate or alveolar ridge. Analysis included documentation of clinical inflammation, polymerase chain reaction, and culture detection of A. actinomycetemcomitans and microcomputed tomography quantitation of peri-implant bone volume. RESULTS Viable A. actinomycetemcomitans biofilm was successfully established on titanium implants in vitro, detected by confocal laser scanning microscopy. An inflammatory response characterized by clinical inflammation, bleeding, ulceration, hyperplasia, and necrosis was observed around biofilm-inoculated implants. A. actinomycetemcomitans was detected by polymerase chain reaction and culture analysis on 100% of biofilm-inoculated implants for up to 3 weeks and 25% for up to 6 weeks. Microcomputed tomography analysis demonstrated significantly lower bone volume (P <0.05) around biofilm-inoculated implants (29.6% ± 7.6%) compared to non-inoculated implants (50.5% ± 9.6%) after 6 weeks. CONCLUSIONS These results describe a novel animal model where A. actinomycetemcomitans biofilm was established in vitro on titanium implants before placement in rat oral cavity, leading to an inflammatory response, osteolysis, and tissue destruction. This model may have potential use for investigation of host responses to biofilm pathogens and antibiofilm therapy.

Vesicle-independent extracellular release of a proinflammatory outer membrane lipoprotein in free-soluble form

BackgroundAggregatibacter actinomycetemcomitans is an oral bacterium associated with aggressively progressing periodontitis. Extracellular release of bacterial outer membrane proteins has been suggested to mainly occur via outer membrane vesicles. This study investigated the presence and conservation of peptidoglycan-associated lipoprotein (AaPAL) among A. actinomycetemcomitans strains, the immunostimulatory effect of AaPAL, and whether live cells release this structural outer membrane lipoprotein in free-soluble form independent of vesicles.ResultsThe pal locus and its gene product were confirmed in clinical A. actinomycetemcomitans strains by PCR-restriction fragment length polymorphism and immunoblotting. Culturing under different growth conditions revealed no apparent requirement for the AaPAL expression. Inactivation of pal in a wild-type strain (D7S) and in its spontaneous laboratory variant (D7SS) resulted in pleiotropic cellular effects. In a cell culture insert model (filter pore size 0.02 μm), AaPAL was detected from filtrates when strains D7S and D7SS were incubated in serum or broth in the inserts. Electron microscopy showed that A. actinomycetemcomitans vesicles (0.05–0.2 μm) were larger than the filter pores and that there were no vesicles in the filtrates. The filtrates were immunoblot negative for a cytoplasmic marker, cyclic AMP (cAMP) receptor protein. An ex vivo model indicated cytokine production from human whole blood stimulated by AaPAL.ConclusionFree-soluble AaPAL can be extracellularly released in a process independent of vesicles.

Hierarchical gene expression profiles of HUVEC stimulated by different lipid A structures obtained from Porphyromonas gingivalis and Escherichia coli

The ability of lipid A structural variants to elicit unique endothelial cell gene expression was examined by measuring global gene expression profiles in human umbilical cord vein endothelial cells (HUVEC) using Affymetrix full genome chips. Two lipid A structural variants obtained from Porphyromonas gingivalis designated PgLPS1435/1449 and PgLPS1690 as well as LPS obtained from Escherichia coli wild type and an E. coli msbB mutant (missing myristic acid in the lipid A) were examined. Each of these lipid A structures has been shown to interact with TLR4; however, PgLPS1435/1449 and E. coli msbB LPS have been shown to be TLR4 antagonists while PgLPS1690 and wild‐type E. coli LPS are TLR4 agonists. It was found that PgLPS1435/1449 and PgLPS1690 as well as E. coli msbB LPS activated a subset of those genes significantly transcribed in response to E. coli wild‐type LPS. Furthermore, the subset of genes expressed in response to the different lipid A structural forms were those most significantly activated by wild‐type E. coli LPS demonstrating a hierarchy in TLR4‐dependent endothelial cell gene activation. A unique gene expression profile for the weak TLR4 agonist PgLPS1690 was observed and represents a TLR4 hierarchy in endothelial cell gene activation.

Evolutionary Divergence of Aggregatibacter actinomycetemcomitans

Gram-negative facultative Aggregatibacter actinomycetemcomitans is an oral pathogen associated with periodontitis. The genetic heterogeneity among A. actinomycetemcomitans strains has been long recognized. This study provides a comprehensive genomic analysis of A. actinomycetemcomitans and the closely related nonpathogenic Aggregatibacter aphrophilus. Whole genome sequencing by Illumina MiSeq platform was performed for 31 A. actinomycetemcomitans and 2 A. aphrophilus strains. Sequence similarity analysis shows a total of 3,220 unique genes across the 2 species, where 1,550 are core genes present in all genomes and 1,670 are variable genes (accessory genes) missing in at least 1 genome. Phylogenetic analysis based on 397 concatenated core genes distinguished A. aphrophilus and A. actinomycetemcomitans. The latter was in turn divided into 5 clades: clade b (serotype b), clade c (serotype c), clade e/f (serotypes e and f), clade a/d (serotypes a and d), and clade e′ (serotype e strains). Accessory genes accounted for 14.1% to 23.2% of the A. actinomycetemcomitans genomes, with a majority belonging to the category of poorly characterized by Cluster of Orthologous Groups classification. These accessory genes were often organized into genomic islands (n = 387) with base composition biases, suggesting their acquisitions via horizontal gene transfer. There was a greater degree of similarity in gene content and genomic islands among strains within clades than between clades. Strains of clade e′ isolated from human were found to be missing the genomic island that carries genes encoding cytolethal distending toxins. Taken together, the results suggest a pattern of sequential divergence, starting from the separation of A. aphrophilus and A. actinomycetemcomitans through gain and loss of genes and ending with the divergence of the latter species into distinct clades and serotypes. With differing constellations of genes, the A. actinomycetemcomitans clades may have evolved distinct adaptation strategies to the human oral cavity.

Markedly different genome arrangements between serotype a strains and serotypes b or c strains of Aggregatibacter actinomycetemcomitans

BackgroundBacterial phenotype may be profoundly affected by the physical arrangement of their genes in the genome. The Gram-negative species Aggregatibacter actinomycetemcomitans is a major etiologic agent of human periodontitis. Individual clonal types of A. actinomycetemcomitans may exhibit variable virulence and different patterns of disease association. This study examined the genome arrangement of A. actinomycetemcomitans using the genome sequences of serotypes a-c strains. The genome alignment and rearrangement were analyzed by the MAUVE and the GRIMM algorithms. The distribution patterns of genes along the leading/lagging strands were investigated. The occurrence and the location of repeat sequences relative to the genome rearrangement breakpoints were also determined.ResultsThe genome arrangement of the serotype a strain D7S-1 is markedly different from the serotype b strain HK1651 or the serotype c strain D11S-1. Specific genome arrangements appear to be conserved among strains of the same serotypes. The reversal distance between D7S-1 and HK1651 by GRIMM analysis is also higher than the within-species comparisons of 7 randomly selected bacterial species. The locations of the orthologous genes are largely preserved between HK1651 and D11S-1 but not between D7S-1 and HK1651 (or D11S-1), irrespective of whether the genes are categorized as essential/nonessential or highly/nonhighly expressed. However, genome rearrangement did not disrupt the operons of the A. actinomycetemcomitans strains. A higher proportion of the genome in strain D7S-1 is occupied by repeat sequences than in strains HK1651 or D11S-1.ConclusionThe results suggest a significant evolutionary divergence between serotype a strains and serotypes b/c strains of A. actinomycetemcomitans. The distinct patterns of genome arrangement may suggest phenotypic differences between serotype a and serotypes b/c strains.