Valentina Godovikova

Dentin-pulp complex responses to carious lesions.

To understand the molecular events underlying the dentin-pulp complex responses to carious progression, we systematically analyzed tissue morphology and dentin matrix protein distribution in non-carious teeth and in teeth with enamel and dentin caries. Dentin matrix proteins analyzed included collagen type I, phosphophoryn (PP) and dentin sialoprotein (DSP), all of which play decisive roles in the dentin mineralization process. Human non-carious and carious third molar teeth were freshly collected, demineralized, and processed for hematoxylin and eosin staining. The ABC-peroxidase method was used for immunohistochemical staining of collagen type I, PP and DSP proteins using specific antibodies. In situ hybridization was also performed. In contrast to elongated odontoblasts in non-carious teeth, odontoblasts subjacent to dentin caries were cuboidal and fewer in number. The predentin zone was also dramatically reduced in teeth with dentin caries. The staining intensity for collagen type I, PP and DSP in the dentin-pulp complex increased progressively from non-carious teeth, to teeth with enamel and dentin caries. In situ hybridization studies showed DSP-PP mRNA expression in odontoblasts and dental pulp that was consistent with our immunohistochemical results. These results suggest that carious lesions stimulate the dentin-pulp complex to actively synthesize collagen type I, PP and DSP proteins. This response to carious lesions is likely to provide a basis for reparative and/or reactionary dentin formation.

Dynamic Processing of Recombinant Dentin Sialoprotein-Phosphophoryn Protein

Dentin sialoprotein (DSP) and phosphophoryn (PP) are the two noncollagenous proteins classically linked to dentin but more recently found in bone, kidney, and salivary glands. These two proteins are derived from a single copy DSP-PP gene. Although this suggests that the DSP-PP gene is first transcribed into DSP-PP mRNAs, which later undergo processing to yield the DSP and PP proteins, this mechanism has not yet been demonstrated because of the inability to identify a DSP-PP precursor protein from any cell or tissue sample. To study this problem, we utilized a baculovirus expression system to produce recombinant DSP-PP precursor proteins from a DSP-PP240 cDNA, which represents one of several endogenous DSP-PP transcripts that influence various tooth mineralization phases. Our in vitro results demonstrate that DSP-PP240 precursor proteins are produced by this system and are capable of self-processing to yield both DSP and PP proteins. We further demonstrated that purified recombinant DSP-PP240, purified recombinant PP240, and the native highly phosphorylated protein (equivalent to the PP523 isoform) have proteolytic activity. These newly identified tissue proteases may play key roles in tissue modeling during organogenesis.

Composition and Localization of Treponema denticola Outer Membrane Complexes

ABSTRACT The Treponema denticola outer membrane lipoprotein-protease complex (dentilisin) contributes to periodontal disease by degrading extracellular matrix components and disrupting intercellular host signaling pathways. We recently demonstrated that prcB, located upstream of and cotranscribed with prcA and prtP, encodes a 22-kDa lipoprotein that interacts with PrtP and is required for its activity. Here we further characterize products of the protease locus and their roles in expression, formation, and localization of outer membrane complexes. PrcB migrates in native gels as part of a >400-kDa complex that includes PrtP and PrcA, as well as the major outer sheath protein Msp. PrcB is detectable as a minor constituent of the purified active protease complex, which was previously reported to consist of only PrtP and auxiliary polypeptides PrcA1 and PrcA2. Though it lacks the canonical ribosome binding site present upstream of both prcA and prtP, PrcB is present at levels similar to those of PrtP in whole-cell extracts. Immunofluorescence microscopy demonstrated cell surface exposure of the mature forms of PrtP, PrcA1, PrcB, and Msp. The 16-kDa N-terminal acylated fragment of PrtP (predicted to be released during activation of PrtP) was present in cell extracts but was detected neither in the purified active protease complex nor on the cell surface. PrcA2, detectable on the surface of Msp-deficient cells but not that of wild-type cells, coimmunoprecipitated with Msp. Our results indicate that PrcB is a component of the outer membrane lipoprotein protease complex and that Msp and PrcA2 interaction may mediate formation of a very-high-molecular-weight outer membrane complex.

Treponema denticola upregulates MMP-2 activation in periodontal ligament cells: interplay between epigenetics and periodontal infection.

OBJECTIVE Periodontal pathogens initiate chronic dysregulation of inflammation and tissue homeostasis that characterize periodontal disease. To better understand oral microbe-host tissue interactions, we investigated expression and activation of MMP-2 in periodontal ligament cells following Treponema denticola challenge. DESIGN Cultured PDL cells were challenged with T. denticola, and bacterial adherence, internalization and survival were assayed by immunofluorescence microscopy and antibiotic protection assays, respectively. MMP-2 activation was detected by zymography. MMP-2, MT1/MMP and TIMP-2 expression following T. denticola challenge was determined by qRT-PCR. Promoter methylation of MMP-2 and MT1/MMP was screened by methylation-sensitive restriction analysis and by bisulfite DNA sequencing. RESULTS T. denticola adhered to and was internalized by PDL cells but did not survive intracellularly beyond 24h. Importantly, while dentilisin activity in PDL culture supernatants gradually decreased following T. denticola challenge, MMP-2 activation persisted for up to 5 days, suggesting involvement of other regulatory mechanisms. Transcription and expression of MT1/MMP and TIMP-2 increased in response to T. denticola challenge. However, consistent with previously reported constitutive pro-MMP-2 expression in PDL cells, the MMP-2 promoter was hypomethylated, independent of T. denticola challenge. CONCLUSIONS MMP-2 promoter hypomethylation is consistent with constitutive pro-MMP-2 expression in PDL cells. This, coupled with T. denticola-mediated upregulation of MMP-2-related genes and chronic activation of pro-MMP-2, mimics key in vivo mechanisms of periodontal disease chronicity, in particular MMP-2-dependent matrix degradation and bone resorption. Adherence and/or internalization of T. denticola may contribute to these processes by one or more regulatory mechanisms, including contact-dependent signal transduction or other epigenetic mechanisms.

A Modified Shuttle Plasmid Facilitates Expression of a Flavin Mononucleotide-Based Fluorescent Protein in Treponema denticola ATCC 35405

ABSTRACT Oral pathogens, including Treponema denticola, initiate the dysregulation of tissue homeostasis that characterizes periodontitis. However, progress of research on the roles of T. denticola in microbe-host interactions and signaling, microbial communities, microbial physiology, and molecular evolution has been hampered by limitations in genetic methodologies. This is typified by an extremely low transformation efficiency and inability to transform the most widely studied T. denticola strain with shuttle plasmids. Previous studies have suggested that robust restriction-modification (R-M) systems in T. denticola contributed to these problems. To facilitate further molecular genetic analysis of T. denticola behavior, we optimized existing protocols such that shuttle plasmid transformation efficiency was increased by >100-fold over prior reports. Here, we report routine transformation of T. denticola ATCC 35405 with shuttle plasmids, independently of both plasmid methylation status and activity of the type II restriction endonuclease encoded by TDE0911. To validate the utility of this methodological advance, we demonstrated expression and activity in T. denticola of a flavin mononucleotide-based fluorescent protein (FbFP) that is active under anoxic conditions. Addition of routine plasmid-based fluorescence labeling to the Treponema toolset will enable more-rigorous and -detailed studies of the behavior of this organism.

Treponema denticola increases MMP-2 expression and activation in the periodontium via reversible DNA and histone modifications

Host‐derived matrix metalloproteinases (MMPs) and bacterial proteases mediate destruction of extracellular matrices and supporting alveolar bone in periodontitis. The Treponema denticola dentilisin protease induces MMP‐2 expression and activation in periodontal ligament (PDL) cells, and dentilisin‐mediated activation of pro‐MMP‐2 is required for cellular fibronectin degradation. Here, we report that T. denticola regulates MMP‐2 expression through epigenetic modifications in the periodontium. PDL cells were treated with epigenetic enzyme inhibitors before or after T. denticola challenge. Fibronectin fragmentation, MMP‐2 expression, and activation were assessed by immunoblot, zymography, and qRT‐PCR, respectively. Chromatin modification enzyme expression in T. denticola‐challenged PDL cells and periodontal tissues were evaluated using gene arrays. Several classes of epigenetic enzymes showed significant alterations in transcription in diseased tissue and T. denticola‐challenged PDL cells. T. denticola‐mediated MMP‐2 expression and activation were significantly reduced in PDL cells treated with inhibitors of aurora kinases and histone deacetylases. In contrast, DNA methyltransferase inhibitors had little effect, and inhibitors of histone acetyltransferases, methyltransferases, and demethylases exacerbated T. denticola‐mediated MMP‐2 expression and activation. Chronic epigenetic changes in periodontal tissues mediated by T. denticola or other oral microbes may contribute to the limited success of conventional treatment of chronic periodontitis and may be amenable to therapeutic reversal.

Activation of the Innate Immune System by Treponema denticola Periplasmic Flagella through Toll-Like Receptor 2

ABSTRACT Treponema denticola is an indigenous oral spirochete that inhabits the gingival sulcus or periodontal pocket. Increased numbers of oral treponemes within this environment are associated with localized periodontal inflammation, and they are also part of an anaerobic polymicrobial consortium responsible for endodontic infections. Previous studies have indicated that T. denticola stimulates the innate immune system through Toll-like receptor 2 (TLR2); however, the pathogen-associated molecular patterns (PAMPs) responsible for T. denticola activation of the innate immune system are currently not well defined. In this study, we investigated the role played by T. denticola periplasmic flagella (PF), unique motility organelles of spirochetes, in stimulating an innate immune response. Wild-type T. denticola stimulated the production of the cytokines tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), IL-6, IL-10, and IL-12 by monocytes from human peripheral blood mononuclear cells, while its isogenic nonmotile mutant lacking PF resulted in significantly diminished cytokine stimulation. In addition, highly purified PF were able to dose dependently stimulate cytokine TNF-α, IL-1β, IL-6, IL-10, and IL-12 production in human monocytes. Wild-type T. denticola and the purified PF triggered activation of NF-κB through TLR2, as determined using a variety of TLR-transfected human embryonic 293 cell lines, while the PF-deficient mutants lacked the ability to stimulate, and the complemented PF-positive T. denticola strain restored the activation. These findings suggest that T. denticola stimulates the innate immune system in a TLR2-dependent fashion and that PF are a key bacterial component involved in this process.