SummaryTranscriptional profiling, bioinformatics, statistical and ontology tools were used to uncover and dissect genes and pathways of human gingival epithelial cells that are modulated upon interaction with the periodontal pathogens Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis . Consistent with their biological and clinical differences, the common core transcriptional response of epithelial cells to both organisms was very limited, and organismspecific responses predominated. A large number of differentially regulated genes linked to the P53 apoptotic network were found with both organisms, which was consistent with the pro-apoptotic phenotype observed with A. actinomycetemcomitans and antiapoptotic phenotype of P. gingivalis. Furthermore, with A. actinomycetemcomitans , the induction of apoptosis did not appear to be Fas-or TNF a a a amediated. Linkage of specific bacterial components to host pathways and networks provided additional insight into the pathogenic process. Comparison of the transcriptional responses of epithelial cells challenged with parental P. gingivalis or with a mutant of P. gingivalis deficient in production of major fimbriae, which are required for optimal invasion, showed major expression differences that reverberated throughout the host cell transcriptome. In contrast, gene ORF859 in A. actinomycetemcomitans, which may play a role in intracellular homeostasis, had a more subtle effect on the transcriptome. These studies help unravel the complex and dynamic interactions between host epithelial cells and endogenous bacteria that can cause opportunistic infections.

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The SerB protein of Porphyromonas gingivalis is a HAD family serine phosphatase that plays a critical role in entry and survival of the organism in gingival epithelial cells. SerB is secreted by P. gingivalis upon contact with epithelial cells. Here it is shown by microarray analysis that SerB impacts the transcriptional profile of gingival epithelial cells, with pathways involving the actin cytoskeleton and cytokine production among those significantly overpopulated with differentially regulated genes. Consistent with the transcriptional profile, a SerB mutant of P. gingivalis exhibited defective remodeling of actin in epithelial cells. Interaction between gingival epithelial cells and isolated SerB protein resulted in actin rearrangement and an increase in the F/G actin ratio. SerB protein was also required for P. gingivalis to antagonize interleukin-8 accumulation following stimulation of epithelial cells with Fusobacterium nucleatum. SerB is thus capable of modulating host cell signal transduction that impacts the actin cytoskeleton and cytokine production.

SUMMARYTreponema denticola is associated with subgingival biofilms in adult periodontitis and with acute necrotizing ulcerative gingivitis. However, the molecular mechanisms by which T. denticola impacts periodontal inflammation and alveolar bone resorption remain unclear. Here, we examined changes in the host transcriptional profiles during a T. denticola infection using a murine calvarial model of inflammation and bone resorption. T. denticola was injected into the subcutaneous soft tissue over the calvaria of BALB/c mice for 3 days, after which the soft tissues and the calvarial bones were excised. RNA was isolated and analysed for transcript profiling using Murine GeneChip® arrays. Following T. denticola infection, 2905 and 1234 genes in the infected calvarial bones and soft tissues, respectively, were differentially expressed (p ≤ 0.05). Biological pathways significantly impacted by T. denticola infection in calvarial bone and calvarial tissue included leukocyte transendothelial migration, cell adhesion (immune system) molecules, cell cycle, extracellular matrix-receptor interaction, focal adhesion, B-cell receptor signaling and transforming growth factor-β signaling pathways resulting in proinflammatory, chemotactic effects, and T-cell stimulation. In conclusion, localized T. denticola infection differentially induces transcription of a broad array of host genes, the profiles of which differed between inflamed calvarial bone and soft tissues.

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.