P. S. Zilm

Fusobacterium nucleatum supports the growth of Porphyromonas gingivalis in oxygenated and carbon-dioxide-depleted environments.

The authors compared the differences in tolerance to oxygen of the anaerobic periodontopathic bacteria Fusobacterium nucleatum and Porphyromonas gingivalis, and explored the possibility that F. nucleatum might be able to support the growth of P. gingivalis in aerated and CO2-depleted environments. Both micro-organisms were grown as monocultures and in co-culture in the presence and absence of CO2 and under different aerated conditions using a continuous culture system. At steady state, viable counts were performed and the activities of the enzymes superoxide dismutase and NADH oxidase/peroxidase were assayed in P. gingivalis. In co-culture, F. nucleatum was able to support the growth of P. gingivalis in aerated and CO2-depleted environments in which P. gingivalis, as a monoculture, was not able to survive. F. nucleatum not only appeared to have a much higher tolerance to oxygen than P. gingivalis, but a significant increase in its numbers occurred under moderately oxygenated conditions. F. nucleatum might have an additional indirect role in dental plaque maturation, contributing to the reducing conditions necessary for the survival of P. gingivalis and possibly other anaerobes less tolerant to oxygen. Additionally, F. nucleatum is able to generate a capnophilic environment essential for the growth of P. gingivalis.

Proteomic characterization of mesenchymal stem cell-like populations derived from ovine periodontal ligament, dental pulp, and bone marrow: analysis of differentially expressed proteins.

Postnatal mesenchymal stem/stromal-like cells (MSCs) including periodontal ligament stem cells (PDLSCs), dental pulp stem cells (DPSCs), and bone marrow stromal cells (BMSCs) are capable of self-renewal and differentiation into multiple mesenchymal cell lineages. Despite their similar expression of MSC-associated and osteoblastic markers, MSCs retain the capacity to generate structures resembling the microenvironments from which they are derived in vivo and represent a promising therapy for the regeneration of complex tissues in the clinical setting. With this in mind, systematic approaches are required to identify the differential protein expression patterns responsible for lineage commitment and mediating the formation of these complex structures. This is the first study to compare the differential proteomic expression profiles of ex vivo-expanded ovine PDLSCs, DPSCs, and BMSCs derived from an individual donor. The two-dimensional electrophoresis was performed and regulated proteins were identified by liquid chromatography--electrospray-ionization tandem mass spectrometry (MS and MS/MS), database searching, and de novo sequencing. In total, 58 proteins were differentially expressed between at least 2 MSC populations in both sheep, 12 of which were up-regulated in one MSC population relative to the other two. In addition, the regulation of selected proteins was also conserved between equivalent human MSC populations. We anticipate that differential protein expression profiling will provide a basis for elucidating the protein expression patterns and molecular cues that are crucial in specifying the characteristic growth and developmental capacity of dental and non-dental tissue-derived MSC populations. These expression patterns can serve as important tools for the regeneration of particular tissues in future stem cell-based tissue engineering studies using animal models.

Dengue Virus Infection Induces Upregulation of GRP78, Which Acts To Chaperone Viral Antigen Production

ABSTRACT Dengue virus (DENV) pathogenesis is related to the host responses to viral infection within target cells, and therefore, this study assessed intracellular changes in host proteins following DENV infection. Two-dimensional gel electrophoresis and mass spectrometry identified upregulation of the host endoplasmic reticulum (ER) chaperone GRP78 in K562 cells following DENV infection, in the absence of virus-induced cell death. Upregulation of GRP78 in DENV-infected cells was confirmed by immunostaining and confocal microscopy and by Western blot analysis and was also observed in DENV-infected primary monocyte-derived macrophages, a natural target cell type for DENV infection. GRP78 was upregulated in both DENV antigen-positive and -negative cells in the DENV-infected culture, suggesting a bystander effect, with the highest GRP78 levels coincident with high-level DENV antigen production and infectious-virus release. Transfection of target cells to express GRP78 prior to DENV challenge did not affect subsequent DENV infection, but cleavage of GRP78 with the SubAB toxin, during an established DENV infection, yielded a 10- to 100-fold decrease in infectious-virus release, loss of intracellular DENV particles, and a dramatic decrease in intracellular DENV antigen. However, DENV RNA levels were unchanged, indicating normal DENV RNA replication but altered DENV antigen levels in the absence of GRP78. Thus, GRP78 is upregulated by DENV infection and is necessary for DENV antigen production and/or accumulation. This may be a common requirement for viruses such as flaviviruses that depend heavily on the ER for coordinated protein production and processing.

Sodium Ion-Driven Serine/Threonine Transport in Porphyromonas gingivalis

Porphyromonas gingivalis is an asaccharolytic, gram-negative bacterium that relies on the fermentation of amino acids for metabolic energy. When grown in continuous culture in complex medium containing 4 mM (each) free serine, threonine, and arginine, P. gingivalis assimilated mainly glutamate/glutamine, serine, threonine, aspartate/asparagine, and leucine in free and/or peptide form. Serine and threonine were assimilated in approximately equal amounts in free and peptide form. We characterized serine transport in this bacterium by measuring uptake of the radiolabeled amino acid in washed cells of P. gingivalis energized with a tetrapeptide not containing serine. Serine was transported by a single system with an affinity constant for transport (K(t)) of 24 microM that was competitively inhibited by threonine. Serine transport was dependent on sodium ion concentration in the suspending buffer, and the addition of the ionophore gramicidin caused the inhibition of serine uptake. Together these data indicate that serine transport was sodium ion-motive force driven. A P. gingivalis gene potentially encoding a serine transporter was identified by sequence similarity to an Escherichia coli serine transporter (SstT). This P. gingivalis gene, designated sstT, was inactivated by insertion of a Bacteroides tetQ gene, producing the mutant W50ST. The mutant was unable to transport serine, confirming the presence of a single serine transporter in this bacterium under these growth conditions. The transport of serine by P. gingivalis was dependent on the presence of free cysteine in the suspension buffer. Other reducing agents were unable to stimulate serine uptake. These data show that P. gingivalis assimilates free serine and threonine from culture media via a cysteine-activated, sodium ion-motive force-driven serine/threonine transporter.

Effect of dietary omega-3 polyunsaturated fatty acids on experimental periodontitis in the mouse.

BACKGROUND AND OBJECTIVE Periodontitis is an infective disease caused predominantly by gram-negative anerobes. The host inflammatory response to these bacteria causes alveolar bone loss, which characterizes periodontitis. Omega-3 polyunsaturated fatty acids have recognized anti-inflammatory effects; their oxygenated derivatives are key mediators in reducing inflammation. In this study we tested the hypothesis that dietary supplementation with tuna fish oil rich in the n-3 polyunsaturated fatty acid, docosahexaenoic acid, would reduce alveolar bone loss in mice inoculated with periodontopathic bacteria. MATERIAL AND METHODS Adult mice were fed experimental diets containing either 10% tuna oil or Sunola oil for 57 d. After 14 d, 35 mice on each diet were inoculated orally with Porphyromonas gingivalis, with a mixture of P. gingivalis and Fusobacterium nucleatum, with carboxymethylcellulose or remained untreated. The mice were killed, and soft tissue biopsies from the oral cavity of treated mice were used to determine the polyunsaturated fatty acid concentrations. The maxilla was removed, stained and digitally imaged to assess bone loss around the upper molars. RESULTS n-3 polyunsaturated fatty acid levels were significantly higher in oral soft tissues of mice fed tuna oil compared with the control group. Mice fed tuna oil and inoculated with P. gingivalis or with the combination of F. nucleatum and P. gingivalis exhibited 72% and 54% less alveolar bone loss respectively, compared with the treatment control group. CONCLUSION Alveolar bone loss was inversely related to n-3 polyunsaturated fatty acid tissue levels. In conclusion, fish oil dietary supplementation may have potential benefits as a host modulatory agent in the prevention and/or adjunctive management of periodontitis.

The inability of Streptococcus mutans and Lactobacillus acidophilus to form a biofilm in vitro on dentine pretreated with ozone

BACKGROUND The use of ozone therapy in the treatment of dental caries is equivocal. The aim of this study was to use an in vitro model to determine the effects of prior ozone application to dentine on biofilm formation and to measure any associated reduction in bacteria viability. METHODS Twenty dentine discs were bonded to the bases of 5 mL polycarbonate screw top vials. Ten dentine discs were infused with ozone for 40 seconds, 10 samples remained untreated as a control. The vials were filled with nutrient medium, sterilized and placed into the outflow from a continuous chemostat culture of Streptococcus mutans and Lactobacillus acidophilus for four weeks. At the conclusion of the experiment bacterial growth was monitored by taking optical density readings of the growth medium in each vial and the outer surface of the dentine specimens were examined by scanning electron microscopy as shown by SEM analysis. RESULTS Ozone infusion prevented biofilm formation on all the treated samples while there was substantial biofilm present on the control specimens. While the average optical density of the control specimens was almost twice that of the ozone infused dentine (0.710 for the control with a SD of 0.288 and 0.446 for the ozonated samples with a SD of 0.371), the results were not significant (p > 0.05). CONCLUSIONS This preliminary study has shown that the infusion of ozone into non-carious dentine prevented biofilm formation in vitro from S. mutans and L. acidophilus over a four-week period. The possibility exists that ozone treatment may alter the surface wettability of dentine through reaction with organic constituents.

'Chocolate' silver nanoparticles: Synthesis, antibacterial activity and cytotoxicity.

HYPOTHESIS Silver nanoparticles (AgNPs) have emerged as a powerful weapon against antibiotic resistant microorganisms. However, most conventional AgNPs syntheses require the use of hazardous chemicals and generate toxic organic waste. Hence, in recent years, plant derived and biomolecule based synthetics have has gained much attention. Cacao has been used for years for its medicinal benefits and contains a powerful reducing agent - oxalic acid. We hypothesized that, due to the presence of oxalic acid, cacao extract is capable of reducing silver nitrate (AgNO3) to produce AgNPs. EXPERIMENTS In this study, AgNPs were synthesized by using natural cacao extract as a reducing and stabilizing agent. The reaction temperature, time and reactant molarity were varied to optimize the synthesis yield. FINDINGS UV-visible spectroscopy (UV-vis), dynamic light scattering (DLS) and transmission electron microscopy (TEM) characterization demonstrated that the synthesized AgNPs were spherical particles ranging in size from 35 to 42.5nm. The synthesized AgNPs showed significant antibacterial activity against clinically relevant pathogens such as Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Staphylococcus epidermidis. Importantly, these green AgNPs are not cytotoxic to human dermal fibroblasts (HDFs) at concentrations below 32μg/ml. We conclude that cacao-based synthesis is a reproducible and sustainable method for the generation of stable antimicrobial silver nanoparticles with low cytotoxicity to human cells. The AgNPs synthesized in this work have promising properties for applications in the biomedical field.

Influence of arginine on the coexistence ofStreptococcus mutans andS. milleri in glucose-limited mixed continuous culture

Dental plaque is a complex community of bacteria coexisting in an environment frequently limited by carbon and energy sources. UnlikeStreptococcus mutans, other oral streptococci such asS. milleri andS. sanguis have an absolute requirement for and actually consume all available arginine when grown glucose limited in a chemically defined medium. The conditions, particularly in terms of arginine concentration, under which the dental plaque bacteriaS. mutans andS. milleri would coexist under glucose-limiting conditions were investigated. The minimum level of arginine supporting optimal growth ofS. milleri was found to be ca. 50μM, and above this level these strains outcompetedS. mutans. However, coexistence withS. mutans could be achieved at arginine levels of 14–40μM, depending upon theS. milleri andS. mutans strains used. Under such dual limitation,S. milleri was unable to respond to glucose pulses but did respond to pulses of arginine and arginine plus glucose. One of the twoS. milleri strains did not tolerate low pH. In contrast,S. mutans did not tolerate high pH whereasS. milleri was unaffected. This is relevant to dental plaque where arginine catabolism produces a pH rise. Additionally, arginine is an important nutrient since it can be used as an energy source by some oral streptococci.

Proteomic identification of proteinase inhibitors in the porcine enamel matrix derivative, EMD®

BACKGROUND AND OBJECTIVE The porcine enamel matrix derivative, EMD(®), which is the active component of Emdogain(®), is used widely in periodontics because of its ability to promote the regeneration of soft and hard tissues and to reduce inflammation. Previous studies have used indirect methods to explain its angiogenic and proliferative effects on cells associated with wound healing. In this study we used proteomic techniques to identify proteins in EMD other than amelogenins. MATERIAL AND METHODS Proteins in EMD were separated by two-dimensional gel electrophoresis and were identified using mass spectrometry. Proteomic results were validated by western blot analysis of Emdogain. RESULTS Fourteen proteins of porcine origin were identified and included the serine and cysteine proteinase inhibitors alpha1-antichymotrypsin and fetuin A, respectively. Alpha1-antichymotrypsin is an acute-phase factor that has been reported to indirectly down-regulate the expression of the gelatinase MMP-9. Fetuin A, a major glycoprotein component of bone and teeth, is a potent inhibitor of ectopic calcification of vascular and soft tissues and has been implicated in both osteogenesis and bone resorption. It also facilitates plasma membrane repair in damaged fibroblasts. CONCLUSION EMD contains a number of high-molecular-weight compounds which include the proteinase inhibitors, fetuin A and alpha1-antichymotrypsin.

A proteomic investigation of Fusobacterium nucleatum alkaline-induced biofilms

BackgroundThe Gram negative anaerobe Fusobacterium nucleatum has been implicated in the aetiology of periodontal diseases. Although frequently isolated from healthy dental plaque, its numbers and proportion increase in plaque associated with disease. One of the significant physico-chemical changes in the diseased gingival sulcus is increased environmental pH. When grown under controlled conditions in our laboratory, F. nucleatum subspecies polymorphum formed mono-culture biofilms when cultured at pH 8.2. Biofilm formation is a survival strategy for bacteria, often associated with altered physiology and increased virulence. A proteomic approach was used to understand the phenotypic changes in F. nucleatum cells associated with alkaline induced biofilms. The proteomic based identification of significantly altered proteins was verified where possible using additional methods including quantitative real-time PCR (qRT-PCR), enzyme assay, acidic end-product analysis, intracellular polyglucose assay and Western blotting.ResultsOf 421 proteins detected on two-dimensional electrophoresis gels, spot densities of 54 proteins varied significantly (p < 0.05) in F. nucleatum cultured at pH 8.2 compared to growth at pH 7.4. Proteins that were differentially produced in biofilm cells were associated with the functional classes; metabolic enzymes, transport, stress response and hypothetical proteins. Our results suggest that biofilm cells were more metabolically efficient than planktonic cells as changes to amino acid and glucose metabolism generated additional energy needed for survival in a sub-optimal environment. The intracellular concentration of stress response proteins including heat shock protein GroEL and recombinational protein RecA increased markedly in the alkaline environment. A significant finding was the increased abundance of an adhesin, Fusobacterial outer membrane protein A (FomA). This surface protein is known for its capacity to bind to a vast number of bacterial species and human epithelial cells and its increased abundance was associated with biofilm formation.ConclusionThis investigation identified a number of proteins that were significantly altered by F. nucleatum in response to alkaline conditions similar to those reported in diseased periodontal pockets. The results provide insight into the adaptive mechanisms used by F. nucleatum biofilms in response to pH increase in the host environment.