Enhanced transformation efficiency in Treponema denticola enabled by SyngenicDNA-based plasmids lacking restriction-modification target motifs.

Oral spirochetes are among a small group of keystone pathogens contributing to dysregulation of tissue homeostatic processes that leads to breakdown of the tissue and bone supporting the teeth in periodontal disease. Additionally, our group has recently demonstrated that Treponema are among the dominant microbial genera detected intracellularly in tumor specimens from patients with oral squamous cell carcinoma. While over 60 species and phylotypes of oral Treponema have been detected, T. denticola is one of the few that can be grown in culture and the only one in which genetic manipulation is regularly performed. Thus, T. denticola is a key model organism for studying spirochete metabolic processes, interactions with other microbes, and host cell and tissue responses relevant to oral diseases, as well as venereal and nonvenereal treponematoses whose agents lack workable genetic systems. We previously demonstrated improved transformation efficiency using an Escherichia coli-T. denticola shuttle plasmid and its utility for expression in T. denticola of an exogenous fluorescent protein that is active under anaerobic conditions. Here, we expand on this work by characterizing T. denticola Type I and Type II restriction-modification (R-M) systems and designing a high-efficiency R-M-silent "SyngenicDNA" shuttle plasmid resistant to all T. denticola ATCC 35405 R-M systems. Resequencing of the ATCC 33520 genome revealed an additional Type I R-M system consistent with the relatively low transformation efficiency of the shuttle plasmid in this strain. Using SyngenicDNA approaches, we optimized shuttle plasmid transformation efficiency in T. denticola and used it to complement a defined T. denticola ΔfhbB mutant strain. We further report the first high-efficiency transposon mutagenesis of T. denticola using an R-M-silent, codon-optimized, himarC9 transposase-based plasmid. Thus, use of SyngenicDNA-based strategies and tools can enable further mechanistic examinations of T. denticola physiology and behavior.

Targeting Cariogenic Streptococcus mutans in Oral Biofilms with Charge-Switching Smart Antimicrobial Polymers.

Cariogenic biofilms produce strong acidic microenvironments, which is the primary cause of dental caries. Streptococcus mutans is a dominant species in cariogenic biofilms. Herein, we report a pH-responsive, charge-switching smart copolymer to selectively target and eradicate bacteria in cariogenic biofilms. To that end, the copolymer is designed to be activated in an acidic environment. The smart copolymer, Poly-1A, consists of ternary compositions of monomers with a cationic ethyl ammonium group, a carboxylic group, and a hydrophobic group in the side chains. The net charge of Poly-1A was charge neutral at neutral pH, but it switched to be cationic because the acidic carboxylate side chains were protonated and became neutral; however, the ammonium groups remained positive. Poly-1A with a net positive charge bound to the anionic surface of oral bacteria by electrostatic interactions and disrupted the bacterial membranes, causing bacterial death. Poly-1A reduced the cell viability of planktonic and biofilm S. mutans at pH 4.5, while it was not bactericidal at pH 7.4. Poly-1A did not reduce the cell viability of human gingival fibroblasts and periodontal ligament stem cells for a 1 h incubation.

Characterization of Treponema denticola Major Surface Protein (Msp) by Deletion Analysis and Advanced Molecular Modeling.

Treponema denticola, a keystone pathogen in periodontitis, is a model organism for studying Treponema physiology and host-microbe interactions. Its major surface protein Msp forms an oligomeric outer membrane complex that binds fibronectin, has cytotoxic pore-forming activity, and disrupts several intracellular processes in host cells. T. denticola msp is an ortholog of the Treponema pallidum tprA to -K gene family that includes tprK, whose remarkable in vivo hypervariability is proposed to contribute to T. pallidum immune evasion. We recently identified the primary Msp surface-exposed epitope and proposed a model of the Msp protein as a ß-barrel protein similar to Gram-negative bacterial porins. Here, we report fine-scale Msp mutagenesis demonstrating that both the N and C termini as well as the centrally located Msp surface epitope are required for native Msp oligomer expression. Removal of as few as three C-terminal amino acids abrogated Msp detection on the T. denticola cell surface, and deletion of four residues resulted in complete loss of detectable Msp. Substitution of a FLAG tag for either residues 6 to 13 of mature Msp or an 8-residue portion of the central Msp surface epitope resulted in expression of full-length Msp but absence of the oligomer, suggesting roles for both domains in oligomer formation. Consistent with previously reported Msp N-glycosylation, proteinase K treatment of intact cells released a 25 kDa polypeptide containing the Msp surface epitope into culture supernatants. Molecular modeling of Msp using novel metagenome-derived multiple sequence alignment (MSA) algorithms supports the hypothesis that Msp is a large-diameter, trimeric outer membrane porin-like protein whose potential transport substrate remains to be identified. IMPORTANCE The Treponema denticola gene encoding its major surface protein (Msp) is an ortholog of the T. pallidum tprA to -K gene family that includes tprK, whose remarkable in vivo hypervariability is proposed to contribute to T. pallidum immune evasion. Using a combined strategy of fine-scale mutagenesis and advanced predictive molecular modeling, we characterized the Msp protein and present a high-confidence model of its structure as an oligomer embedded in the outer membrane. This work adds to knowledge of Msp-like proteins in oral treponemes and may contribute to understanding the evolutionary and potential functional relationships between T. denticola Msp and the orthologous T. pallidum Tpr proteins.

Intracerebral but Not Peripheral Infection of Live Porphyromonas gingivalis Exacerbates Alzheimer's Disease Like Amyloid Pathology in APP-TgCRND8 Mice.

The impact of oral microbial dysbiosis on Alzheimer's disease (AD) remains controversial. Building off recent studies reporting that various microbes might directly seed or promote amyloid ß (Aß) deposition, we evaluated the effects of periodontal bacteria (Porphyromonas gingivalis, Treponema denticola) and supragingival commensal (Streptococcus gordonii) oral bacterial infection in the APP-transgenic CRND8 (Tg) mice model of AD. We tracked bacterial colonization and dissemination, and monitored effects on gliosis and amyloid deposition. Chronic oral infection did not accelerate Aß deposition in Tg mice but did induce alveolar bone resorption, IgG immune response, and an intracerebral astrogliosis (GFAP: glial fibrillary acidic protein). In contrast, intracerebral inoculation of live but not heat-killed P. gingivalis increased Aß deposition and Iba-1 (ionized calcium-binding adaptor-1) microgliosis after 8 weeks of bacterial infection but not at 4 days. These data show that there may be differential effects of infectious microbes on glial activation and amyloid deposition depending on the species and route of inoculation, and thereby provide an important framework for future studies. Indeed, these studies demonstrate marked effects on amyloid ß deposition only in a fairly non-physiologic setting where live bacteria is injected directly into the brain.

Approaches to Understanding Mechanisms of Dentilisin Protease Complex Expression in Treponema denticola.

The oral spirochete Treponema denticola is a keystone periodontal pathogen that, in association with members of a complex polymicrobial oral biofilm, contributes to tissue damage and alveolar bone loss in periodontal disease. Virulence-associated behaviors attributed to T. denticola include disruption of the host cell extracellular matrix, tissue penetration and disruption of host cell membranes accompanied by dysregulation of host immunoregulatory factors. T. denticola dentilisin is associated with several of these behaviors. Dentilisin is an outer membrane-associated complex of acylated subtilisin-family PrtP protease and two other lipoproteins, PrcB and PrcA, that are unique to oral spirochetes. Dentilisin is encoded in a single operon consisting of prcB-prcA-prtP. We employ multiple approaches to study mechanisms of dentilisin assembly and PrtP protease activity. To determine the role of each protein in the protease complex, we have made targeted mutations throughout the protease locus, including polar and nonpolar mutations in each gene (prcB, prcA, prtP) and deletions of specific PrtP domains, including single base mutagenesis of key PrtP residues. These will facilitate distinguishing between host cell responses to dentilisin protease activity and its acyl groups. The boundaries of the divergent promoter region and the relationship between dentilisin and the adjacent iron transport operon are being resolved by incremental deletions in the sequence immediately 5' to the protease locus. Comparison of the predicted three-dimensional structure of PrtP to that of other subtilisin-like proteases shows a unique PrtP C-terminal domain of approximately 250 residues. A survey of global gene expression in the presence or absence of protease gene expression reveals potential links between dentilisin and iron uptake and homeostasis in T. denticola. Understanding the mechanisms of dentilisin transport, assembly and activity of this unique protease complex may lead to more effective prophylactic or therapeutic treatments for periodontal disease.

Immunotopological Analysis of the Treponema denticola Major Surface Protein (Msp).

Treponema denticola, one of several recognized periodontal pathogens, is a model organism for studying Treponema physiology and host-microbe interactions. Its major surface protein Msp (or MOSP) comprises an oligomeric outer membrane-associated complex that binds fibronectin, has cytotoxic pore-forming activity, and disrupts several intracellular responses. There are two hypotheses regarding native Msp structure and membrane topology. One hypothesis predicts that the entire Msp protein forms a ß-barrel structure similar to that of well-studied outer membrane porins of Gram-negative bacteria. The second hypothesis predicts a bipartite Msp with distinct and separate periplasmic N-terminal and porin-like ß-barrel C-terminal domains. The bipartite model, based on bioinformatic analysis of the orthologous Treponema pallidum Tpr proteins, is supported largely by studies of recombinant TprC and Msp polypeptides. The present study reports immunological studies in both T. denticola and Escherichia coli backgrounds to identify a prominent Msp surface epitope (residues 229 to 251 in ATCC 35405) in a domain that differs between strains with otherwise highly conserved Msps. These results were then used to evaluate a series of in silico structural models of representative T. denticola Msps. The data presented here are consistent with a model of Msp as a large-diameter ß-barrel porin. This work adds to the knowledge regarding the diverse Msp-like proteins in oral treponemes and may contribute to an understanding of the evolutionary and potential functional relationships between Msps of oral Treponema and the orthologous group of Tpr proteins of T. pallidum.IMPORTANCETreponema denticola is among a small subset of the oral microbiota contributing to severe periodontal disease. Due to its relative genetic tractability, T. denticola is a model organism for studying Treponema physiology and host-microbe interactions. T. denticola Msp is a highly expressed outer membrane-associated oligomeric protein that binds fibronectin, has cytotoxic pore-forming activity, and disrupts intracellular regulatory pathways. It shares homology with the orthologous group of T. pallidum Tpr proteins, one of which is implicated in T. pallidum in vivo antigenic variation. The outer membrane topologies of both Msp and the Tpr family proteins are unresolved, with conflicting reports on protein domain localization and function. In this study, we combined empirical immunological data derived both from diverse T. denticola strains and from recombinant Msp expression in E. coli with in silico predictive structural modeling of T. denticola Msp membrane topology, to move toward resolution of this important issue in Treponema biology.

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

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-1ß (IL-1ß), 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-1ß, 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.

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.

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

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 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.

Composition and localization of Treponema denticola outer membrane complexes.

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 PrcB is required for expression and activity of the PrcA-PrtP (dentilisin) complex.

The Treponema denticola surface protease complex, consisting of PrtP protease (dentilisin) and two auxiliary polypeptides (PrcA1 and PrcA2), is believed to contribute to periodontal disease by degrading extracellular matrix components and disrupting host intercellular signaling. Previously, we showed that transcription of the protease operon initiates upstream of TDE0760 (herein designated prcB), the open reading frame immediately 5' of prcA-prtP. The prcB gene is conserved in T. denticola strains. PrcB localizes to the detergent phase of Triton X-114 cell surface extracts and migrates as a 22-kDa polypeptide, in contrast to the predicted 17-kDa cytoplasmic protein encoded in the annotated T. denticola genome. Consistent with this observation, the PrcB N terminus is unavailable for Edman sequencing, suggesting that it is acylated. Nonpolar deletion of prcB in T. denticola showed that PrcB is required for production of PrtP protease activity, including native PrtP cleavage of PrcA to PrcA1 and PrcA2. A 6xHis-tagged PrcB protein coimmunoprecipitates with native PrtP, using either anti-PrtP or anti-His-tag antibodies, and recombinant PrtP copurifies with PrcB-6xHis in nickel affinity chromatography. Taken together, these data are consistent with identification of PrcB as a PrtP-binding lipoprotein that likely stabilizes the PrtP polypeptide during localization to the outer membrane.

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-PP(240) cDNA, which represents one of several endogenous DSP-PP transcripts that influence various tooth mineralization phases. Our in vitro results demonstrate that DSP-PP(240) 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-PP(240), purified recombinant PP(240), and the native highly phosphorylated protein (equivalent to the PP(523) isoform) have proteolytic activity. These newly identified tissue proteases may play key roles in tissue modeling during organogenesis.

A rat 8 kb dentin sialoprotein-phosphophoryn (DSP-PP) promoter directs spatial and temporal LacZ activity in mouse tissues.

Dentin sialoprotein (DSP) and phosphophoryn (PP) are two major dentin noncollagenous proteins that are encoded on a single DSP-PP transcript whose expression is tightly regulated during tooth dentinogenesis. The recent identification of this gene transcript in other tissues, including inner ear and jaw tissue, suggests that DSP and PP may have pleiotropic effects on other organs besides teeth. To identify candidate regulatory elements that control DSP-PP temporal and spatial expression, we constructed a -5 kb upstream region rat DSP-PP promoter into the beta-galactosidase expression vector pnLacF plasmid and used this construct to prepare DSP-PP-LacZ transgenic mice. Multiple mouse tissues including teeth, bone, and kidney obtained from the six resulting transgenic mouse lines displayed strong LacZ activity. This spatial distribution was confirmed in several of these tissues by in situ hybridization studies. LacZ activity was transiently expressed in preameloblasts and continuously expressed in odontoblasts demonstrating that this -5 kb rat promoter-dependent LacZ expression mimics reported DSP-PP mRNA expression patterns. Interestingly, this -5 kb rat promoter construct drives LacZ expression according to the rat developmental clock. Based on identified transcription factors present in this -5 kb promoter region, we have identified several probable cis-regulatory modules whose interaction with one another could account for the spatial and temporal distribution of DSP-PP transcripts in developing tissues.