Time-lapse confocal microscopy to study in vitro Streptococcus mutans surface colonization.

The cariogenicity of Streptococcus mutans relates to its ability to form biofilms on dental surfaces. The aim of this work was to develop a flowcell system compatible with time-lapse confocal microscopy to compare the adhesion and accumulation of S. mutans cells on surfaces in unsupplemented media against media containing sucrose or sucralose (a non-metabolized sweetener) over a short period of time. Fluorescent S. mutans 3209/pVMCherry was suspended in unsupplemented media or media supplemented with 1% sucrose or 1% sucralose and passed through a 3D-printed flowcell system. Flowcells were imaged over 60 minutes using a confocal microscope. Image analysis was performed, including a newly developed object-movement-based method to measure biomass adhesion. Streptococcus mutans 3209/pVMCherry grown in 1% sucrose-supplemented media formed small, dense, relatively immobile clumps in the flowcell system measured by biovolume, surface area, and median object centroid movement. Sucralose-supplemented and un-supplemented media yielded large, loose, mobile aggregates. Architectural metrics and per-object movement were significantly different (P < 0.05) when comparing sucrose-supplemented media to either unsupplemented or sucralose-supplemented media. These results demonstrate the utility of a flowcell system compatible with time-lapse confocal microscopy and image analysis when studying initial biofilm formation and adhesion under different nutritional conditions.

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.

Natural monoterpenes-laden electrospun fibrous scaffolds for endodontic infection eradication.

This investigation aimed to synthesize poly(D,L-lactide) (PLA)-based fibrous scaffolds containing natural essential oils (i.e., linalool and citral) and determine their antimicrobial properties and cytocompatibility as a clinically viable cell-friendly disinfection strategy for regenerative endodontics. PLA-based fibrous scaffolds were fabricated via electrospinning with different concentrations of linalool and citral. The micromorphology and average diameter of the fibers was investigated through scanning electron microscopy (SEM). The chemical composition of the scaffolds was inferred by Fourier-transform infrared spectroscopy (FTIR). Antimicrobial efficacy against Enterococcus faecalis and Actinomyces naeslundii was also evaluated by agar diffusion and colony-forming units (CFU) assays. The scaffolds' cytocompatibility was determined using dental pulp stem cells (DPSCs). Statistical analyses were performed and the significance level was set at α = 5%. Linalool and citral's incorporation in the PLA fibrous scaffolds was confirmed in the FTIR spectra. SEM images indicate no morphological changes upon inclusion of the essential oils, except the reduced diameter of 40% linalool-laden fibers (p < 0.05). Importantly, significant antimicrobial properties were reported for citral-containing scaffolds for CFU/mL counts (p < 0.05), while only 20% and 40% linalool-laden scaffolds reduced CFU/mL (p < 0.05). Meanwhile, the inhibition halos were verified in a concentration-dependent manner for all monoterpenes-laden scaffolds. Citral- and linalool-laden PLA-based fibrous scaffolds showed acceptable cytocompatibility. The incorporation of natural monoterpenes did not alter the scaffolds' fibrous morphology, promoted antimicrobial action against endodontic pathogens, and preserved DPSCs viability. Linalool- and citral-laden electrospun scaffolds hold promise as naturally derived antimicrobial therapeutics for applications in regenerative endodontics.

Hypochlorous acid inactivates oral pathogens and a SARS-CoV-2-surrogate.

BACKGROUND: Droplets and aerosols produced during dental procedures are a risk factor for microbial and viral transmission. Unlike sodium hypochlorite, hypochlorous acid (HOCl) is nontoxic to tissues but still exhibits broad microbicidal effect. HOCl solution may be applicable as a supplement to water and/or mouthwash. This study aims to evaluate the effectiveness of HOCl solution on common human oral pathogens and a SARS-CoV-2 surrogate MHV A59 virus, considering the dental practice environment. METHODS: HOCl was generated by electrolysis of 3% hydrochloric acid. The effect of HOCl on human oral pathogens, Fusobacterium nucleatum, Prevotella intermedia, Streptococcus intermedius, Parvimonas micra, and MHV A59 virus was studied from four perspectives: concentration; volume; presence of saliva; and storage. HOCl solution in different conditions was utilized in bactericidal and virucidal assays, and the minimum inhibitory volume ratio that is required to completely inhibit the pathogens was determined. RESULTS: In the absence of saliva, the minimum inhibitory volume ratio of freshly prepared HOCl solution (45-60 ppm) was 4:1 for bacterial suspensions and 6:1 for viral suspensions. The presence of saliva increased the minimum inhibitory volume ratio to 8:1 and 7:1 for bacteria and viruses, respectively. Applying a higher concentration of HOCl solution (220 or 330 ppm) did not lead to a significant decrease in the minimum inhibitory volume ratio against S. intermedius and P. micra. The minimum inhibitory volume ratio increases in applications of HOCl solution via the dental unit water line. One week of storage of HOCl solution degraded HOCl and increased the minimum growth inhibition volume ratio. CONCLUSIONS: HOCl solution (45-60 ppm) is still effective against oral pathogens and SAR-CoV-2 surrogate viruses even in the presence of saliva and after passing through the dental unit water line. This study indicates that the HOCl solution can be used as therapeutic water or mouthwash and may ultimately reduce the risk of airborne infection in dental practice.

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.

Periodontal pathogens promote cancer aggressivity via TLR/MyD88 triggered activation of Integrin/FAK signaling that is therapeutically reversible by a probiotic bacteriocin.

Epidemiological studies reveal significant associations between periodontitis and oral cancer. However, knowledge about the contribution of periodontal pathogens to oral cancer and potential regulatory mechanisms involved is limited. Previously, we showed that nisin, a bacteriocin and commonly used food preservative, reduced oral cancer tumorigenesis and extended the life expectancy in tumor-bearing mice. In addition, nisin has antimicrobial effects on key periodontal pathogens. Thus, the purpose of this study was to test the hypothesis that key periodontal pathogens (Porphyromonas gingivalis, Treponema denticola, and Fusobacterium nucleatum) promote oral cancer via specific host-bacterial interactions, and that bacteriocin/nisin therapy may modulate these responses. All three periodontal pathogens enhanced oral squamous cell carcinoma (OSCC) cell migration, invasion, tumorsphere formation, and tumorigenesis in vivo, without significantly affecting cell proliferation or apoptosis. In contrast, oral commensal bacteria did not affect OSCC cell migration. Pathogen-enhanced OSCC cell migration was mediated via integrin alpha V and FAK activation, since stably blocking alpha V or FAK expression abrogated these effects. Nisin inhibited these pathogen-mediated processes. Further, Treponema denticola induced TLR2 and 4 and MyD88 expression. Stable suppression of MyD88 significantly inhibited Treponema denticola-induced FAK activation and abrogated pathogen-induced migration. Together, these data demonstrate that periodontal pathogens contribute to a highly aggressive cancer phenotype via crosstalk between TLR/MyD88 and integrin/FAK signaling. Nisin can modulate these pathogen-mediated effects, and thus has therapeutic potential as an antimicrobial and anti-tumorigenic agent.

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.

Electrospun Azithromycin-Laden Gelatin Methacryloyl Fibers for Endodontic Infection Control.

This study was aimed at engineering photocrosslinkable azithromycin (AZ)-laden gelatin methacryloyl fibers via electrospinning to serve as a localized and biodegradable drug delivery system for endodontic infection control. AZ at three distinct amounts was mixed with solubilized gelatin methacryloyl and the photoinitiator to obtain the following fibers: GelMA+5%AZ, GelMA+10%AZ, and GelMA+15%AZ. Fiber morphology, diameter, AZ incorporation, mechanical properties, degradation profile, and antimicrobial action against Aggregatibacter actinomycetemcomitans and Actinomyces naeslundii were also studied. In vitro compatibility with human-derived dental pulp stem cells and inflammatory response in vivo using a subcutaneous rat model were also determined. A bead-free fibrous microstructure with interconnected pores was observed for all groups. GelMA and GelMA+10%AZ had the highest fiber diameter means. The tensile strength of the GelMA-based fibers was reduced upon AZ addition. A similar pattern was observed for the degradation profile in vitro. GelMA+15%AZ fibers led to the highest bacterial inhibition. The presence of AZ, regardless of the concentration, did not pose significant toxicity. In vivo findings indicated higher blood vessel formation, mild inflammation, and mature and thick well-oriented collagen fibers interweaving with the engineered fibers. Altogether, AZ-laden photocrosslinkable GelMA fibers had adequate mechanical and degradation properties, with 15%AZ displaying significant antimicrobial activity without compromising biocompatibility.

Association between metabolic syndrome and periodontitis: The role of lipids, inflammatory cytokines, altered host response, and the microbiome.

Periodontitis has been associated with many systemic diseases and conditions, including metabolic syndrome. Metabolic syndrome is a cluster of conditions that occur concomitantly and together they increase the risk of cardiovascular disease and double the risk of type 2 diabetes. In this review, we focus on the association between metabolic syndrome and periodontitis; however, we also include information on diabetes mellitus and cardiovascular disease, since these two conditions are significantly intertwined with metabolic syndrome. With regard to periodontitis and metabolic syndrome, to date, the vast majority of studies point to an association between these two conditions and also demonstrate that periodontitis can contribute to the development of, or can worsen, metabolic syndrome. Evaluating the effect of metabolic syndrome on the salivary microbiome, data presented herein support the hypothesis that the salivary bacterial profile is altered in metabolic syndrome patients compared with healthy patients. Considering periodontitis and these three conditions, the vast majority of human and animal studies point to an association between periodontitis and metabolic syndrome, diabetes, and cardiovascular disease. Moreover, there is evidence to suggest that metabolic syndrome and diabetes can alter the oral microbiome. However, more studies are needed to fully understand the influence these conditions have on each other.

Roles of TroA and TroR in Metalloregulated Growth and Gene Expression in Treponema denticola.

The availability of divalent metal cations required as cofactors for microbial metabolism is severely limited in the host environment. Bacteria have evolved highly regulated uptake systems to maintain essential metal homeostasis to meet cellular demands while preventing toxicity. The Tro operon (troABCDR), present in all sequenced Treponema spp., is a member of a highly conserved family of ATP-binding cassette transporters involved in metal cation uptake whose expression is controlled by TroR, a DtxR-like cation-responsive regulatory protein. Transcription of troA responds to divalent manganese and iron (T. denticola) or manganese and zinc (T. pallidum), and metal-dependent TroR binding to the troA promoter represses troA transcription. We report here the construction and complementation of defined T. denticola ΔtroR and ΔtroA strains to characterize (i) the role of TroA in metal-dependent T. denticola growth and (ii) the role of TroR in T. denticola gene expression. We show that TroA expression is required for T. denticola growth under iron- and manganese-limited conditions. Furthermore, TroR is required for the transcriptional regulation of troA in response to iron or manganese, and deletion of troR results in significant differential expression of more than 800 T. denticola genes in addition to troA These results suggest that (i) TroA-mediated cation uptake is important in metal homeostasis in vitro and may be important for Treponema survival in the host environment and (ii) the absence of TroR results in significant dysregulation of nearly one-third of the T. denticola genome. These effects may be direct (as with troA) or indirect due to dysregulation of metal homeostasis.IMPORTANCETreponema denticola is one of numerous host-associated spirochetes, a group including commensals, pathobionts, and at least one frank pathogen. While most T. denticola research concerns its role in periodontitis, its relative tractability for growth and genetic manipulation make it a useful model for studying Treponema physiology, metabolism, and host-microbe interactions. Metal micronutrient acquisition and homeostasis are highly regulated both in microbial cells and by host innate defense mechanisms that severely limit metal cation bioavailability. Here, we characterized the T. denticolatroABCDR operon, the role of TroA-mediated iron and manganese uptake in growth, and the effects of TroR on global gene expression. This study contributes to our understanding of the mechanisms involved in cellular metal homeostasis required for survival in the host environment.

Hybrid Antimicrobial Hydrogel as Injectable Therapeutics for Oral Infection Ablation.

Oral bacterial infection represents the leading cause of the gradual destruction of tooth and periodontal structures anchoring the teeth. Lately, injectable hydrogels have gained increased attention as a promising minimally invasive platform for localized delivery of personalized therapeutics. Here, an injectable and photocrosslinkable gelatin methacryloyl (GelMA) hydrogel is successfully engineered with ciprofloxacin (CIP)-eluting short nanofibers for oral infection ablation. For this purpose, CIP or its ß-cyclodextrin (ß-CD)-inclusion complex (CIP/ß-CD-IC) has been incorporated into polymeric electrospun fibers, which were subsequently cut into short nanofibers, and then embedded in GelMA to obtain an injectable hybrid antimicrobial hydrogel. Thanks to the solubility enhancement of CIP by ß-CD-IC and the tunable degradation profile of GelMA, the hydrogels promote localized, sustained, and yet effective cell-friendly antibiotic doses, as measured by a series of bacterial assays that demonstrated efficacy in attenuating the growth of Gram-positive Enterococcus faecalis. Altogether, we foresee significant potential in translating this innovative hybrid hydrogel as an injectable platform technology that may have broad applications in oral infection ablation, such as periodontal disease and pulpal pathology.

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.

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.

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.

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.

Impact of surface characteristics on the peri-implant microbiome in health and disease.

BACKGROUND: Because little is known about the impact of implant surface modifications on the peri-implant microbiome, we aimed to examine peri-implant communities in various surface types in order to better understand the impact of these surfaces on the development of peri-implantitis (PI). METHODS: One hundred and six systemically healthy individuals with anodized (AN), hydroxyapatite-coated (HA), or sandblasted acid-etched (SLA) implants that were >6 months in function were recruited and categorized into health (H) or PI. Peri-implant biofilm was analyzed using 16S rRNA gene sequencing and compared between health/disease and HA/SLA/AN using community-level and taxa-level metrics. RESULTS: Healthy implants did not demonstrate significant differences in clustering, alpha- or beta-diversity based on surface modification. AN and HA surfaces displayed significant differences between health and PI (p < 0.05); however, such a clustering was not evident with SLA (p > 0.05). AN and HA surfaces also differed in the magnitude and diversity of differences between health and PI. Six species belonging to the genera Shuttleworthia, Scardovia, and Prevotella demonstrated lower abundances in AN implants with PI, and 18 species belonging to the genera Fretibacterium, Tannerella, Treponema, and Fusobacterium were elevated, while in HA implants with PI, 20 species belonging to the genera Streptococcus, Lactobacillus, Veillonella, Rothia, and family Ruminococcaceae were depleted and Peptostreptococcaceae, Atopobiaceae, Veillonellaceae, Porphyromonadaceae, Desulfobulbaceae, and order Synergistales were enriched. CONCLUSIONS: Within the limitations of this study, we demonstrate that implant surface can differentially modify the disease-associated microbiome, suggesting that surface topography must be considered in the multi-factorial etiology of peri-implant diseases.

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).

Multifunctional and biodegradable methacrylated gelatin/Aloe vera nanofibers for endodontic disinfection and immunomodulation.

Currently employed approaches and materials used for vital pulp therapies (VPTs) and regenerative endodontic procedures (REPs) lack the efficacy to predictably achieve successful outcomes due to their inability to achieve adequate disinfection and/or lack of desired immune modulatory effects. Natural polymers and medicinal herbs are biocompatible, biodegradable, and present several therapeutic benefits and immune-modulatory properties; thus, standing out as a clinically viable approach capable of establishing a conducive environment devoid of bacteria and inflammation to support continued root development, dentinal bridge formation, and dental pulp tissue regeneration. However, the low stability and poor mechanical properties of the natural compounds have limited their application as potential biomaterials for endodontic procedures. In this study, Aloe vera (AV), as a natural antimicrobial and anti-inflammatory agent, was incorporated into photocrosslinkable Gelatin methacrylate (GelMA) nanofibers with the purpose of developing a highly biocompatible biomaterial capable of eradicating endodontic infection and modulating inflammation. Stable GelMA/AV nanofibers with optimal properties were obtained at the ratio of (70:30) by electrospinning. In addition to the pronounced antibacterial effect against Enterococcus faecalis, the GelMA/AV (70:30) nanofibers also exhibited a sustained antibacterial activity over 14 days and significant biofilm reduction with minimal cytotoxicity, as well as anti-inflammatory properties and immunomodulatory effects favoring healing. Our results indicate that the novel GelMA/AV (70:30) nanofibers hold great potential as a biomaterial strategy for endodontic infection eradication and enhanced healing.

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.