Purification of Native Dentilisin Complex from Treponema denticola by Preparative Continuous Polyacrylamide Gel Electrophoresis and Functional Analysis by Gelatin Zymography.

Periodontal disease is characterized by the destruction of the hard and soft tissues comprising the periodontium. This destruction translates to a degradation of the extracellular matrices (ECM), mediated by bacterial proteases, host-derived matrix metalloproteinases (MMPs), and other proteases released by host tissues and immune cells. Bacterial pathogens interact with host tissue, triggering adverse cellular functions, including a heightened immune response, tissue destruction, and tissue migration. The oral spirochete Treponema denticola is highly associated with periodontal disease. Dentilisin, a T. denticola outer membrane protein complex, contributes to the chronic activation of pro-MMP-2 in periodontal ligament (PDL) cells and triggers increased expression levels of activators and effectors of active MMP-2 in PDL cells. Despite these advances, no mechanism for dentilisin-induced MMP-2 activation or PDL cytopathic behaviors leading to disease is known. Here, we describe a method for purification of large amounts of the dentilisin protease complex from T. denticola and demonstrate its ability to activate MMP-2, a key regulator of periodontal tissue homeostasis. The T. denticola dentilisin and MMP-2 activation model presented here may provide new insights into the dentilisin protein and identify potential therapeutic targets for further research. Key features • This protocol builds upon a method described by Cunningham et al. [1] for selective release of Treponema outer membrane proteins. • We adapted the protocol for the purification of biologically active, detergent-stable outer membrane protein complexes from large batch cultures of T. denticola. • The protocol involves large-scale preparative electrophoresis using a Model 491 Prep Cell. • We then use gelatin zymography to demonstrate the activity of the purified dentilisin complex by its ability to activate matrix metalloproteinase 2 (MMP-2).

Stimulation of Human Periodontal Ligament Fibroblasts Using Purified Dentilisin Extracted from Treponema denticola.

Periodontal disease is a chronic multifactorial disease triggered by a complex of bacterial species. These interact with host tissues to cause the release of a broad array of pro-inflammatory cytokines, chemokines, and tissue remodelers, such as matrix metalloproteinases (MMPs), which lead to the destruction of periodontal tissues. Patients with severe forms of periodontitis are left with a persistent pro-inflammatory transcriptional profile throughout the periodontium, even after clinical intervention, leading to the destruction of teeth-supporting tissues. The oral spirochete, Treponema denticola , is consistently found at significantly elevated levels at sites with advanced periodontal disease. Of all T. denticola virulence factors that have been described, its chymotrypsin-like protease complex, also called dentilisin, has demonstrated a multitude of cytopathic effects consistent with periodontal disease pathogenesis, including alterations in cellular adhesion activity, degradation of various endogenous extracellular matrix-substrates, degradation of host chemokines and cytokines, and ectopic activation of host MMPs. Thus, the following model of T. denticola -human periodontal ligament cell interactions may provide new knowledge about the mechanisms that drive the chronicity of periodontal disease at the protein, transcriptional, and epigenetic levels, which could afford new putative therapeutic targets. This protocol was validated in: PLOS Pathog (2021), DOI: 10.1371/journal.ppat.1009311.

Paradigm shift in the pathogenesis and treatment of oral cancer and other cancers focused on the oralome and antimicrobial-based therapeutics.

The oral microbiome is a community of microorganisms, comprised of bacteria, fungi, viruses, archaea, and protozoa, that form a complex ecosystem within the oral cavity. Although minor perturbations in the environment are frequent and compensable, major shifts in the oral microbiome can promote an unbalanced state, known as dysbiosis. Dysbiosis can promote oral diseases, including periodontitis. In addition, oral dysbiosis has been associated with other systemic diseases, including cancer. The objective of this review is to evaluate the epidemiologic evidence linking periodontitis to oral, gastrointestinal, lung, breast, prostate, and uterine cancers, as well as describe new evidence and insights into the role of oral dysbiosis in the etiology and pathogenesis of the cancer types discussed. Finally, we discuss how antimicrobials, antimicrobial peptides, and probiotics may be promising tools to prevent and treat these cancers, targeting both the microbes and associated carcinogenesis processes. These findings represent a novel paradigm in the pathogenesis and treatment of cancer focused on the oral microbiome and antimicrobial-based therapies.

Treponema denticola dentilisin triggered TLR2/MyD88 activation upregulates a tissue destructive program involving MMPs via Sp1 in human oral cells.

Periodontal disease is driven by dysbiosis in the oral microbiome, resulting in over-representation of species that induce the release of pro-inflammatory cytokines, chemokines, and tissue-remodeling matrix metalloproteinases (MMPs) in the periodontium. These chronic tissue-destructive inflammatory responses result in gradual loss of tooth-supporting alveolar bone. The oral spirochete Treponema denticola, is consistently found at significantly elevated levels in periodontal lesions. Host-expressed Toll-Like Receptor 2 (TLR2) senses a variety of bacterial ligands, including acylated lipopolysaccharides and lipoproteins. T. denticola dentilisin, a surface-expressed protease complex comprised of three lipoproteins has been implicated as a virulence factor in periodontal disease, primarily due to its proteolytic activity. While the role of acylated bacterial components in induction of inflammation is well-studied, little attention has been given to the potential role of the acylated nature of dentilisin. The purpose of this study was to test the hypothesis that T. denticola dentilisin activates a TLR2-dependent mechanism, leading to upregulation of tissue-destructive genes in periodontal tissue. RNA-sequencing of periodontal ligament cells challenged with T. denticola bacteria revealed significant upregulation of genes associated with extracellular matrix organization and degradation including potentially tissue-specific inducible MMPs that may play novel roles in modulating host immune responses that have yet to be characterized within the context of oral disease. The Gram-negative oral commensal, Veillonella parvula, failed to upregulate these same MMPs. Dentilisin-induced upregulation of MMPs was mediated via TLR2 and MyD88 activation, since knockdown of expression of either abrogated these effects. Challenge with purified dentilisin upregulated the same MMPs while a dentilisin-deficient T. denticola mutant had no effect. Finally, T. denticola-mediated activation of TLR2/MyD88 lead to the nuclear translocation of the transcription factor Sp1, which was shown to be a critical regulator of all T. denticola-dependent MMP expression. Taken together, these data suggest that T. denticola dentilisin stimulates tissue-destructive cellular processes in a TLR2/MyD88/Sp1-dependent fashion.

Treponema denticola-Induced RASA4 Upregulation Mediates Cytoskeletal Dysfunction and MMP-2 Activity in Periodontal Fibroblasts.

The periodontal complex consists of the periodontal ligament (PDL), alveolar bone, and cementum, which work together to turn mechanical load into biological responses that are responsible for maintaining a homeostatic environment. However oral microbes, under conditions of dysbiosis, may challenge the actin dynamic properties of the PDL in the context of periodontal disease. To study this process, we examined host-microbial interactions in the context of the periodontium via molecular and functional cell assays and showed that human PDL cell interactions with Treponema denticola induce actin depolymerization through a novel actin reorganization signaling mechanism. This actin reorganization mechanism and loss of cell adhesion is a pathological response characterized by an initial upregulation of RASA4 mRNA expression resulting in an increase in matrix metalloproteinase-2 activity. This mechanism is specific to the T. denticola effector protein, dentilisin, thereby uncovering a novel effect for Treponema denticola-mediated RASA4 transcriptional activation and actin depolymerization in primary human PDL cells.

Host-microbe interactions: Profiles in the transcriptome, the proteome, and the metabolome.

Periodontal studies using transcriptomics, proteomics, and metabolomics encompass the collection of mRNA transcripts, proteins, and small-molecule chemicals in the context of periodontal health and disease. The number of studies using these approaches has significantly increased in the last decade and they have provided new insight into the pathogenesis and host-microbe interactions that define periodontal diseases. This review provides an overview of current molecular findings using -omic approaches that underlie periodontal disease, including modulation of the host immune response, tissue homeostasis, and complex metabolic processes of the host and the oral microbiome. Integration of these -omic approaches will broaden our perspective of the molecular mechanisms involved in periodontal disease, advancing and improving the diagnosis and treatment of various stages and forms of periodontal disease.