Selenomonas sputigena Interactions with Gingival Epithelial Cells That Promote Inflammation.

Increased prevalence and abundance of Selenomonas sputigena have been associated with periodontitis, a chronic inflammatory disease of tooth-supporting tissues, for more than 50 years. Over the past decade, molecular surveys of periodontal disease using 16S and shotgun metagenomic sequencing approaches have confirmed the disease association of classically recognized periodontal pathogens such as Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia while highlighting previously underappreciated organisms such as Filifactor alocis and S. sputigena. Despite abundant clinical association between S. sputigena and periodontal disease, we have little to no understanding of its pathogenic potential, and virulence mechanisms have not been studied. In this study, we sought to characterize the response of gingival epithelial cells to infection with S. sputigena. Here, we show that S. sputigena attaches to gingival keratinocytes and induces expression and secretion of cytokines and chemokines associated with inflammation and leukocyte recruitment. We demonstrate that S. sputigena induces signaling through Toll-like receptor 2 (TLR2) and TLR4 but evades activation of TLR5. Cytokines released from S. sputigena-infected keratinocytes induced monocyte and neutrophil chemotaxis. These results show that S. sputigena-host interactions have the potential to contribute to bacterially driven inflammation and tissue destruction, the hallmark of periodontitis. Characterization of previously unstudied pathogens may provide novel approaches to develop therapeutics to treat or prevent periodontal disease.

Treponema denticola Induces Interleukin-36γ Expression in Human Oral Gingival Keratinocytes via the Parallel Activation of NF-κB and Mitogen-Activated Protein Kinase Pathways.

The oral epithelial barrier acts as both a physical barrier to the abundant oral microbiome and a sentry for the immune system that, in health, constrains the accumulation of the polymicrobial plaque biofilm. The immune homeostasis during gingivitis that is largely protective becomes dysregulated, unproductive, and destructive to gingival tissue as periodontal disease progresses to periodontitis. The progression to periodontitis is associated with the dysbiosis of the oral microbiome, with increasing prevalences and abundances of periodontal pathogens such as Treponema denticola. Despite the association of T. denticola with a chronic inflammatory disease, relatively little is known about gingival epithelial cell responses to T. denticola infection. Here, we characterized the transcriptome of gingival keratinocytes following T. denticola challenge and identified interleukin-36γ (IL-36γ) as the most differentially expressed cytokine. IL-36γ expression is regulated by p65 NF-κB and the activation of both the Jun N-terminal protein kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) pathways downstream of Toll-like receptor 2 (TLR2). Finally, we demonstrate for the first time that mitogen- and stress-activated kinase 1 (MSK1) contributes to IL-36γ expression and may link the activation of MAPK and NF-κB signaling. These findings suggest that the interactions of T. denticola with the gingival epithelium lead to elevated IL-36γ expression, which may be a critical inducer and amplifier of gingival inflammation and subsequent alveolar bone loss.

Characterization of c-di-AMP signaling in the periodontal pathobiont, Treponema denticola.

Pathobionts associated with periodontitis, such as Treponema denticola, must possess numerous sensory transduction systems to adapt to the highly dynamic subgingival environment. To date, the signaling pathways utilized by T. denticola to rapidly sense and respond to environmental stimuli are mainly unknown. Bis-(3'-5') cyclic diadenosine monophosphate (c-di-AMP) is a nucleotide secondary messenger that regulates osmolyte transport, central metabolism, biofilm development, and pathogenicity in many bacteria but is uncharacterized in T. denticola. Here, we studied c-di-AMP signaling in T. denticola to understand how it contributes to T. denticola physiology. We demonstrated that T. denticola produces c-di-AMP and identified enzymes that function in the synthesis (TDE1909) and hydrolysis (TDE0027) of c-di-AMP. To investigate how c-di-AMP may impact T. denticola cellular processes, a screening assay was performed to identify putative c-di-AMP receptor proteins. This approach identified TDE0087, annotated as a potassium uptake protein, as the first T. denticola c-di-AMP binding protein. As potassium homeostasis is critical for maintaining turgor pressure, we demonstrated that T. denticola c-di-AMP concentrations are impacted by osmolarity, suggesting that c-di-AMP negatively regulates potassium uptake in hypoosmotic solutions. Collectively, this study demonstrates T. denticola utilizes c-di-AMP signaling, identifies c-di-AMP metabolism proteins, identifies putative receptor proteins, and correlates c-di-AMP signaling to osmoregulation.