NLRP3 inflammasome activity and periodontal disease pathogenesis-A bidirectional relationship.

OBJECTIVE: Periodontitis is an inflammatory oral disease that occurs as a result of the damaging effects of the immune response against the subgingival microflora. Among the mechanisms involved, the nucleotide-binding oligomerization domain, leucine-rich repeat-containing proteins family member NLRP3 (NLR family pyrin domain-containing 3), proposed as the key regulator of macrophage-induced inflammation, is strongly associated with periodontal disease due to the bacterial activators. This paper aimed to present key general concepts of NLRP3 inflammasome activation and regulation in periodontal disease. METHOD: A narrative review was conducted in order to depict the current knowledge on the relationship between NLRP3 inflammasome activity and periodontal disease. In vitro and in situ studies were retrieved and commented based on their relevance in the field. RESULTS: The NLRP3 inflammasome activity stimulated by periodontal microbiota drive periodontal disease pathogenesis and progression. This occurs through the release of proinflammatory cytokines IL-1ß, IL-18, and DAMPs (damage-associated molecular pattern molecules) following inflammasome activation. Moreover, the tissue expression of NLRP3 is dysregulated by oral microbiota, further exacerbating periodontal inflammation. CONCLUSION: The review provides new insights into the relationship between the NLRP3 inflammasome activity and periodontal disease pathogenesis, highlighting the roles and regulatory mechanism of inflammatory molecules involved in the disease process.

Tannerella forsythia scavenges Fusobacterium nucleatum secreted NOD2 stimulatory molecules to dampen oral epithelial cell inflammatory response.

The oral organism Tannerella forsythia is auxotrophic for peptidoglycan amino sugar N-acetylmuramic acid (MurNAc). It survives in the oral cavity by scavenging MurNAc- and MurNAc-linked peptidoglycan fragments (muropeptides) secreted by co-habiting bacteria such as Fusobacterium nucleatum with which it forms synergistic biofilms. Muropeptides, MurNAc-l-Ala-d-isoGln (MDP, muramyl dipeptide) and d-γ-glutamyl-meso-DAP (iE-DAP dipeptide), are strong immunostimulatory molecules that activate nucleotide oligomerization domain (NOD)-like innate immune receptors and induce the expression of inflammatory cytokines and antimicrobial peptides. In this study, we utilized an in vitro T. forsythia-F. nucleatum co-culture model to determine if T. forsythia can selectively scavenge NOD ligands from the environment and impact NOD-mediated inflammation. The results showed that NOD-stimulatory molecules were secreted by F. nucleatum in the spent culture broth, which subsequently induced cytokine and antimicrobial peptide expression in oral epithelial cells. In the spent broth from T. forsythia-F. nucleatum co-cultures, the NOD-stimulatory activity was significantly reduced. These data indicated that F. nucleatum releases NOD2-stimulatory muropeptides in the environment, and T. forsythia can effectively scavenge the muropeptides released by co-habiting bacteria to dampen NOD-mediated host responses. This proof-of-principle study demonstrated that peptidoglycan scavenging by T. forsythia can impact the innate immunity of oral epithelium by dampening NOD activation.

Effect of residue and weed management practices on weed flora, yield, energetics, carbon footprint, economics and soil quality of zero tillage wheat.

A two-year field study was conducted during Rabi 2018-2019 and 2019-20 to find out the influence of different residue and weed management practices on weed dynamics, growth, yield, energetics, carbon footprint, economics and soil properties in zero-tilled sown wheat at Research Farm, AICRP-Weed management, SKUAST-Jammu. The experiment with four rice residue management practices and four weed management practices was conducted in a Strip-Plot Design and replicated thrice. The results showed that residue retention treatments recorded lower weed density, biomass and higher wheat growth, yield attributes and yields of wheat as compared to no residue treatment. The magnitude of increase in wheat grain yield was 17.55, 16.98 and 7.41% when treated with 125% recommended dose of nitrogen + residue + waste decomposer (RDN + R + WD), 125% RDN + R, and 100% RDN + R, respectively, compared to no residue treatment. Further, all three herbicidal treatments decreased weed density and biomass than weedy treatments. Consequently, a reduction of 29.30, 28.00, and 25.70% in grain yield were observed in control as compared to sulfosulfuron + carfentrazone, clodinafop-propargyl + metasulfuron, and clodinafop-propargyl + metribuzin, respectively. Moreover, 125% RDN + R + WD obtained significantly higher energy output (137860 MJ ha-1) and carbon output (4522 kg CE/ha), but 100% RDN had significantly higher net energy (101802 MJ ha-1), energy use efficiency (7.66), energy productivity (0.23 kg MJ-1), energy profitability (6.66 kg MJ-1), carbon efficiency (7.66), and less carbon footprint (7.66) as compared to other treatments. Despite this, treatments with 125% RDN + R + WD and 125% RDN + R provided 17.58 and 16.96% higher gross returns, and 24.45% and 23.17% net outcomes, respectively, than that of control. However, compared to the control, sulfosulfuron + carfentrazone showed considerably higher energy output (140492 MJ ha-1), net energy (104778 MJ ha-1), energy usage efficiency (4.70), energy productivity (0.14 kg MJ-1), energy profitability (3.70 kg MJ-1), carbon output (4624 kg CE ha-1), carbon efficiency (4.71), and lower carbon footprint (0.27). Furthermore, sulfosulfuron + carfentrazone, clodinafop-propargyl + metasulfuron, and clodinafop-propargyl + metribuzin recorded 29.29% and 38.42%, 27.99%, and 36.91%, 25.69% and 34.32% higher gross returns and net returns over control treatment, respectively. All three herbicides showed higher gross returns, net returns, and benefit cost ratio over control. The soil nutrient status was not significantly affected either by residue or weed management practices. Therefore, based on present study it can be concluded that rice residue retention with 25% additional nitrogen and weed management by clodinafop-propargyl + metasulfuron herbicide found suitable for zero tillage wheat.

An Extracytoplasmic Function Sigma/Anti-Sigma Factor System Regulates ß-Glucanase Expression in Tannerella forsythia in Response to Fusobacterium nucleatum Sensing.

The periodontal pathogen Tannerella forsythia expresses a ß-glucanase (TfGlcA) whose expression is induced in response to Fusobacterium nucleatum, a bridge bacterium of the oral cavity. TfGlcA cleaves ß-glucans to release glucose, which can serve as a carbon source for F. nucleatum and other cohabiting organisms. A two-gene cluster encoding a putative extracytoplasmic function (ECF) sigma factor and a FecR-like anti-sigma factor has been recognized upstream of a TfGlcA operon. We characterized and analyzed the role of these putative ECF sigma and anti-sigma factors in the regulation of TfGlcA expression. For this purpose, deletion mutants were constructed and analyzed for ß-glucanase expression. In addition, an Escherichia coli-produced ECF sigma factor recombinant protein was evaluated for transcriptional and DNA binding activities. The results showed that the recombinant protein promoted transcription by the RNA polymerase core enzyme from the glcA promoter. Furthermore, in comparison to those in the parental strain, the ß-glucanase expression levels were significantly reduced in the ECF sigma-factor deletion mutant and increased significantly in the FecR anti-sigma factor deletion mutant. The levels did not change in the mutants following coincubation with the F. nucleatum whole cells or cell extracts. Finally, the levels of ß-glucanase produced by T. forsythia strains paralleled F. nucleatum biomass in cobiofilms. In conclusion, we identified a ß-glucanase operon regulatory system in T. forsythia comprising an ECF sigma factor (TfSigG) and a cognate FecR-like anti-sigma factor responsive to F. nucleatum and potentially other stimuli. IMPORTANCE Previous studies have shown that F. nucleatum forms robust biofilms with T. forsythia utilizing glucose from the hydrolysis of ß-glucans by T. forsythia ß-glucanase, induced by F. nucleatum. In this study, we showed that a regulatory system comprising of an ECF sigma factor, TfSigG, and a FecR-like anti-sigma factor, TfFecR, is responsible for the ß-glucanase induction in response to F. nucleatum, suggesting that this system plays roles in the mutualistic interactions of T. forsythia and F. nucleatum. The findings suggest the development and potential utility of small-molecule inhibitors targeting the ß-glucanase activity or the TfSigG/TfFecR system as therapeutic drugs against dental plaque formation and periodontitis.

Investigating Peptidoglycan Recycling Pathways in Tannerella forsythia with N-Acetylmuramic Acid Bioorthogonal Probes.

The human oral microbiome is the second largest microbial community in humans, harboring over 700 bacterial species, which aid in digestion and protect from growth of disease-causing pathogens. One such oral pathogen, Tannerella forsythia, along with other species, contributes to the pathogenesis of periodontitis. T. forsythia is unable to produce its own N-acetylmuramic acid (NAM) sugar, essential for peptidoglycan biosynthesis and therefore must scavenge NAM from other species with which it cohabitates. Here, we explore the recycling potential of T. forsythia for NAM uptake with a bioorthogonal modification into its peptidoglycan, allowing for click-chemistry-based visualization of the cell wall structure. Additionally, we identified NAM recycling enzyme homologues in T. forsythia that are similar to the enzymes found in Pseudomonas putida. These homologues were then genetically transformed into a laboratory safe Escherichia coli strain, resulting in the efficient incorporation of unnatural NAM analogues into the peptidoglycan backbone and its visualization, alone or in the presence of human macrophages. This strain will be useful in further studies to probe NAM recycling and peptidoglycan scavenging pathways of T. forsythia and other cohabiting bacteria.

Tannerella forsythia strains differentially induce interferon gamma-induced protein 10 (IP-10) expression in macrophages due to lipopolysaccharide heterogeneity.

Tannerella forsythia is strongly implicated in the development of periodontitis, an inflammatory disease that destroys the bone and soft tissues supporting the tooth.  To date, the knowledge of the virulence attributes of T. forsythia species has mainly come from studies with a laboratory adapted strain (ATCC 43037). In this study, we focused on two T. forsythia clinical isolates, UB4 and UB20, in relation to their ability to activate macrophages. We found that these clinical isolates differentially induced proinflammatory cytokine expression in macrophages. Prominently, the expression of the chemokine protein IP-10 (CXCL10) was highly induced by UB20 as compared to UB4 and the laboratory strain ATCC 43037. Our study focused on the lipopolysaccharide component (LPS) of these strains and found that UB20 expressed a smooth-type LPS, unlike UB4 and ATCC 43037 each of which expressed a rough-type LPS. The LPS from UB20, via activation of TLR4, was found to be a highly potent inducer of IP-10 expression via signaling through STAT1 (signal transducer and activator of transcription-1). These data suggest that pathogenicity of T. forsythia species could be strain dependent and the LPS heterogeneity associated with the clinical strains might be responsible for their pathogenic potential and severity of periodontitis.

Porphyromonas gingivalis indirectly elicits intestinal inflammation by altering the gut microbiota and disrupting epithelial barrier function through IL9-producing CD4+ T cells.

Recent epidemiological studies have shown that inflammatory bowel disease is associated with periodontal disease. The oral-gut microbiota axis is a potential mechanism intersecting the two diseases. Porphyromonas gingivalis is currently considered a keystone oral pathogen involved in periodontal disease pathogenesis and disease progression. Recent studies have shown that oral ingestion of P. gingivalis leads to intestinal inflammation. However, the molecular underpinnings of P. gingivalis-mediated gut inflammation have remained elusive. In this study, we show that the oral administration of P. gingivalis indeed leads to ileal inflammation and alteration in gut microbiota with significant reduction in bacterial alpha diversity despite the absence of P. gingivalis in the lower gastrointestinal tract. Utilizing an antibiotic-conditioned mouse model, cecal microbiota transfer experiments were performed to demonstrate that P. gingivalis-induced dysbiotic gut microbiota is sufficient to reproduce gut pathology. Furthermore, we observed a significant expansion in small intestinal lamina propria IL9+ CD4+ T cells, which was negatively correlated with both bacterial and fungal alpha diversity, signifying that P. gingivalis-mediated intestinal inflammation may be due to the subsequent loss of gut microbial diversity. Finally, we detected changes in gene expression related to gut epithelial barrier function, showing the potential downstream effect of intestinal IL9+ CD4+ T-cell induction. This study for the first time showed the mechanism behind P. gingivalis-mediated intestinal inflammation where P. gingivalis indirectly induces intestinal IL9+ CD4+ T cells and inflammation by altering the gut microbiota. Understanding the mechanism of P. gingivalis-mediated intestinal inflammation may lead to the development of novel therapeutic approaches to alleviate the morbidity from inflammatory bowel disease patients with periodontal disease.

B-Cell RANKL Contributes to Pathogen-Induced Alveolar Bone Loss in an Experimental Periodontitis Mouse Model.

Periodontitis is a bacterially-induced inflammatory disease that leads to tooth loss. It results from the damaging effects of a dysregulated immune response, mediated largely by neutrophils, macrophages, T cells and B cells, on the tooth-supporting tissues including the alveolar bone. Specifically, infiltrating B cells at inflamed gingival sites with an ability to secrete RANKL and inflammatory cytokines are thought to play roles in alveolar bone resorption. However, the direct contribution of B cells in alveolar bone resorption has not been fully appreciated. In this study we sought to define the contribution of RANKL expressing B cells in periodontitis by employing a mouse model of pathogen-induced periodontitis that used conditional knockout mice with B cell-targeted RANKL deletion. Briefly, alveolar bone loss was assessed in the wild-type, B-cell deficient (Jh), or B-cell-RANKL deleted (RANKLΔB) mice orally infected with the periodontal pathogen Tannerella forsythia. The RANKLΔB mice were obtained by crossing Cd19-Cre knock-in mice with mice homozygous for conditional RANKL-flox allele (RANKLflox/flox). The alveolar bone resorption was determined by morphometric analysis and osteoclastic activity of the jaw bone. In addition, the bone resorptive potential of the activated effector B cells was assessed ex vivo. The data showed that the RANKL producing B cells increased significantly in the T. forsythia-infected wild-type mice compared to the sham-infected mice. Moreover, T. forsythia-infection induced higher alveolar bone loss in the wild-type and RANKLflox/flox mice compared to infection either in the B cell deficient (Jh) or the B-cell specific RANKL deletion (RANKLΔB) mice. These data established that the oral-pathogen activated B cells contribute significantly to alveolar bone resorption via RANKL production.

Loss of CX3CR1 augments neutrophil infiltration into cochlear tissues after acoustic overstimulation.

The cochlea, the sensory organ for hearing, has a protected immune environment, segregated from the systemic immune system by the blood-labyrinth barrier. Previous studies have revealed that acute acoustic injury causes the infiltration of circulating leukocytes into the cochlea. However, the molecular mechanisms controlling immune cell trafficking are poorly understood. Here, we report the role of CX3CR1 in regulating the entry of neutrophils into the cochlea after acoustic trauma. We employed B6.129P-Cx3cr1tm1Litt /J mice, a transgenic strain that lacks the gene, Cx3cr1, for coding the fractalkine receptor. Our results demonstrate that lack of Cx3cr1 results in the augmentation of neutrophil infiltration into cochlear tissues after exposure to an intense noise of 120 dB SPL for 1 hr. Neutrophil distribution in the cochlea is site specific, and the infiltration level is positively associated with noise intensity. Moreover, neutrophils are short lived and macrophage phagocytosis plays a role in neutrophil clearance, consistent with typical neutrophil dynamics in inflamed non-cochlear tissues. Importantly, our study reveals the potentiation of noise-induced hearing loss and sensory cell loss in Cx3cr1-/- mice. In wild-type control mice (Cx3cr1+/+ ) exposed to the same noise, we also found neutrophils. However, neutrophils were present primarily inside the microvessels of the cochlea, with only a few in the cochlear tissues. Collectively, our data implicate CX3CR1-mediated signaling in controlling neutrophil migration from the circulation into cochlear tissues and provide a better understanding of the impacts of neutrophils on cochlear responses to acoustic injury.

Purification of Tannerella forsythia Surface-Layer (S-Layer) Proteins.

The objective of this chapter is to provide a detailed purification protocol for the surface-layer (S-layer) glycoproteins of the periodontal pathogen Tannerella forsythia. The procedure involves detergent based solubilization of the bacterial S-layer followed by cesium chloride gradient centrifugation and gel permeation chromatography. The protocol is suitable for the isolation of S-layer glycoproteins from T. forsythia strains with diverse O-glycan structures, and aid in understanding the biochemical basis and the role of protein O-glycosylation in bacterial pathogenesis.

New insights on repeated acoustic injury: Augmentation of cochlear susceptibility and inflammatory reaction resultant of prior acoustic injury.

In industrial and military settings, individuals who suffer from one episode of acoustic trauma are likely to sustain another episode of acoustic stress, creating an opportunity for a potential interaction between the two stress conditions. We previously demonstrated that acoustic overstimulation perturbs the cochlear immune environment. However, how the cochlear immune system responds to repeated acoustic overstimulation is unknown. Here, we used a mouse model to investigate the cochlear immune response to repeated stress. We reveal that exposure to an intense noise at 120 dB SPL for 1 h activates the cochlear immune response in a time-dependent fashion with substantial expansion and activation of the macrophage population in the cochlea at 2-days post-exposure. At 20-days post-exposure, the number and pro-inflammatory phenotypes of cochlear macrophages have significantly subsided, but have yet to return to homeostatic levels. Monocytes with anti-inflammatory phenotypes are recruited into the cochlea. With the presence of this residual immune activation, a second exposure to the same noise provokes an exaggerated inflammatory response as evidenced by exacerbated maturation of macrophages. Furthermore, the second noise causes greater sensory cell pathogenesis. Unlike the first noise-induced damage that occurs mainly between 0 and 2 days post-exposure, the second noise-induced damage occurs more frequently between 2 and 20 days post-exposure, the period when secondary damage takes place. These observations suggest that repeated acoustic overstimulation exacerbates cochlear inflammation and secondary sensory cell pathogenesis. Together, our results suggest that the cochlear immune system plays an important role in modulating cochlear responses to repeated acoustic stress.

Persistence of Tannerella forsythia and Fusobacterium nucleatum in dental plaque: a strategic alliance.

Purpose of Review: The Gram-negative oral pathogen Tannerella forsythia is implicated in the pathogenesis of periodontitis, an inflammatory disease characterized by progressive destruction of the tooth supporting structures affecting over 700 million people worldwide. This review highlights the basis of why and how T. forsythia interacts with Fusobacterium nucleatum, a bacterium considered to be a bridge between the early and late colonizing bacteria of the dental plaque. Recent Findings: The recent findings indicate that these two organisms have a strong mutualistic relationship that involves foraging by T. forsythia on F. nucleatum peptidoglycan and utilization of glucose, released by the hydrolytic activity of T. forsythia glucanase, as a nutrient by F. nucleatum. In addition, T. forsythia has the unique ability to generate a toxic and inflammogenic compound, methylglyoxal, from glucose. This compound can induce inflammation, leading to the degradation of periodontal tissues and release of host components as nutrients for bacteria to further exacerbate the disease. Summary: In summary, this article will present our current understanding of mechanisms underpinning T. forsythia-F. nucleatum mutualism, and how this mutualism might impact periodontal disease progression.

Characterization of Porphyromonas gingivalis sialidase and disruption of its role in host-pathogen interactions.

Key to onset and progression of periodontitis is a complex relationship between oral bacteria and the host. The organisms most associated with severe periodontitis are the periodontal pathogens of the red complex: Tannerella forsythia, Treponema denticola and Porphyromonas gingivalis. These organisms express sialidases, which cleave sialic acid from host glycoproteins, and contribute to disease through various mechanisms. Here, we expressed and purified recombinant P. gingivalis sialidase SiaPG (PG_0352) and characterized its activity on a number of substrates, including host sialoglycoproteins and highlighting the inability to cleave diacetylated sialic acids - a phenomenon overcome by the NanS sialate-esterase from T. forsythia. Indeed SiaPG required NanS to maximize sialic acid harvesting from heavily O-acetylated substrates such as bovine salivary mucin, hinting at the possibility of interspecies cooperation in sialic acid release from host sources by these members of the oral microbiota. Activity of SiaPG and P. gingivalis was inhibited using the commercially available chemotherapeutic zanamivir, indicating its potential as a virulence inhibitor, which also inhibited sialic acid release from mucin, and was capable of inhibiting biofilm formation of P. gingivalis on oral glycoprotein sources. Zanamivir also inhibited attachment and invasion of oral epithelial cells by P. gingivalis and other periodontal pathogens, both in monospecies but also in multispecies infection experiments, indicating potential to suppress host-pathogen interactions of a mixed microbial community. This study broadens our understanding of the multifarious roles of bacterial sialidases in virulence, and indicates that their inhibition with chemotherapeutics could be a promising strategy for periodontitis therapy.

Differential fates of tissue macrophages in the cochlea during postnatal development.

The cochlea contains macrophages. These cells participate in inflammatory responses to cochlear pathogenesis. However, it is not clear how and when these cells populate the cochlea during postnatal development. The current study aims to determine the postnatal development of cochlear macrophages with the focus on macrophage development in the organ of Corti and the basilar membrane. Cochleae were collected from C57BL/6J mice at ages of postnatal day (P) 1 to P21, as well as from mature mice (1-4 months). Macrophages were identified based on their expression of F4/80 and Iba1, as well as their unique morphologies. Two sets of macrophages were identified in the regions of the organ of Corti and the basilar membrane. One set resides on the scala tympani side of the basilar membrane. These cells have a round shape at P1 and start to undergo site-specific differentiation at P4. Apical macrophages adopt a dendritic shape. Middle and basal macrophages take on an irregular shape with short projections. Basal macrophages further differentiate into an amoeboid shape. The other set of macrophages resides above the basilar membrane, either beneath the cells of the organ of Corti or along the spiral vessel of the basilar membrane. As the sensory epithelium matures, these cells undergo developmental death with the phenotypes of apoptosis. Macrophages are also identified in the spiral ligament, spiral limbus, and neural regions. Their numbers decrease during postnatal development. Together, these results suggest a dynamic rearrangement of the macrophage population during postnatal cochlear development.

ß-Glucanase Activity of the Oral Bacterium Tannerella forsythia Contributes to the Growth of a Partner Species, Fusobacterium nucleatum, in Cobiofilms.

Tannerella forsythia and Fusobacterium nucleatum are dental plaque bacteria implicated in the development of periodontitis. These two species have been shown to form synergistic biofilms and have been found to be closely associated in dental plaque biofilms. A number of genetic loci for TonB-dependent membrane receptors (TDR) for glycan acquisition, with many existing in association with genes coding for enzymes involved in the breakdown of complex glycans, have been identified in T. forsythia In this study, we focused on a locus, BFO_0186-BFO_0188, that codes for a predicted TDR-SusD transporter along with a putative ß-glucan hydrolyzing enzyme (BFO_0186). This operon is located immediately downstream of a 2-gene operon that codes for a putative stress-responsive extracytoplasmic function (ECF) sigma factor and an anti-sigma factor. Here, we show that BFO_0186 expresses a ß-glucanase that cleaves glucans with ß-1,6 and ß-1,3 linkages. Furthermore, the BFO_0186-BFO_0188 locus is upregulated, with an induction of ß-glucanase activity, in cobiofilms of T. forsythia and F. nucleatum The ß-glucanase activity in mixed biofilms in turn leads to an enhanced hydrolysis of ß-glucans and release of glucose monomers and oligomers as nutrients for F. nucleatum In summary, our study highlights the role of T. forsythia ß-glucanase expressed by the asaccharolytic oral bacterium T. forsythia in the development of T. forsythia-F. nucleatum mixed species biofilms, and suggest that dietary ß-glucans might contribute in plaque development and periodontal disease pathogenesis.IMPORTANCE The development of dental plaque biofilm is a complex process in which metabolic, chemical and physical interactions between bacteria take a central role. Previous studies have shown that the dental pathogens T. forsythia and F. nucleatum form synergistic biofilms and are closely associated in human dental plaque. In this study, we show that ß-glucanase from the periodontal pathogen T. forsythia plays a role in the formation of T. forsythia-F. nucleatum cobiofilms by hydrolyzing ß-glucans to glucose as a nutrient. We also unveiled that the expression of T. forsythia ß-glucanase is induced in response to F. nucleatum sensing. This study highlights the involvement of ß-glucanase activity in the development of T. forsythia-F. nucleatum biofilms and suggests that intake of dietary ß-glucans might be a contributing risk factor in plaque development and periodontal disease pathogenesis.

Regulation and Molecular Basis of Environmental Muropeptide Uptake and Utilization in Fastidious Oral Anaerobe Tannerella forsythia.

Tannerella forsythia is a Gram-negative oral anaerobe associated with periodontitis. This bacterium is auxotrophic for the peptidoglycan amino sugar N-acetylmuramic (MurNAc) and likely relies on scavenging peptidoglycan fragments (muropeptides) released by cohabiting bacteria during their cell wall recycling. Many Gram-negative bacteria utilize an inner membrane permease, AmpG, to transport peptidoglycan fragments into their cytoplasm. In the T. forsythia genome, the Tanf_08365 ORF has been identified as a homolog of AmpG permease. In order to confirm the functionality of Tanf_08365, a reporter system in an Escherichia coli host was generated that could detect AmpG-dependent accumulation of cytosolic muropeptides via a muropeptide-inducible ß-lactamase reporter gene. In trans complementation of this reporter strain with a Tanf_08365 containing plasmid caused significant induction of ß-lactamase activity compared to that with an empty plasmid control. These data indicated that Tanf_08365 acted as a functional muropeptide permease causing accumulation of muropeptides in E. coli and thus suggested that it is a permease involved in muropeptide scavenging in T. forsythia. Furthermore, we showed that the promoter regulating the expression of Tanf_08365 was activated significantly by a hybrid two-component system regulatory protein, GppX. We also showed that compared to the parental T. forsythia strain a mutant lacking GppX in which the expression of AmpG was reduced significantly attenuated in utilizing free muropeptides. In summary, we have uncovered the mechanism by which this nutritionally fastidious microbe accesses released muropeptides in its environment, opening up the possibility of targeting this activity to reduce its numbers in periodontitis patients with potential benefits in the treatment of disease.

Macrophage inducible C-type lectin (Mincle) recognizes glycosylated surface (S)-layer of the periodontal pathogen Tannerella forsythia.

The oral pathogen Tannerella forsythia is implicated in the development of periodontitis, a common inflammatory disease that leads to the destruction of the gum and tooth supporting tissues, often leading to tooth loss. T. forsythia is a unique Gram-negative organism endowed with an elaborate protein O-glycosylation system that allows the bacterium to express a glycosylated surface (S)-layer comprising two high molecular weight glycoproteins modified with O-linked oligosaccharides. The T. forsythia S-layer has been implicated in the modulation of cytokine responses of antigen presenting cells, such as macrophages, that play a significant role during inflammation associated with periodontitis. The macrophage-inducible C-type lectin receptor (Mincle) is an FcRγ-coupled pathogen recognition receptor that recognizes a wide variety of sugar containing ligands from fungal and bacterial pathogens. In this study, we aimed to determine if Mincle might be involved in the recognition of T. forsythia S-layer and modulation of cytokine response of macrophages against the bacterium. Binding studies using recombinant Mincle-Fc fusion protein indicated a specific Ca2+-dependent binding of Mincle to T. forsythia S-layer. Subsequent experiments with Mincle-expressing and Mincle-knockdown macrophages revealed a role for Mincle/S-layer interaction in the induction of both pro- and anti-inflammatory cytokine secretion in macrophages stimulated with T. forsythia as well as its S-layer. Together, these studies revealed Mincle as an important macrophage receptor involved in the modulation of cytokine responses of macrophages against T. forsythia, and thus may play a critical role in orchestrating the host immune response against the bacterium.

Structure of the LPS O-chain from Fusobacterium nucleatum strain 10953, containing sialic acid.

Fusobacterium nucleatum is an anaerobic bacterium found in the human mouth where it causes periodontitis. Recently, it has been gaining attention as a potential causative agent for colorectal cancer and is strongly linked with pregnancy complications including pre-term and still births. Little is known about virulence factors of this organism and thus we have initiated studies to examine the bacterial surface glycochemistry. Consistent with a recent paper suggesting that F. nucleatum strain 10593 can synthesize sialic acid, a staining technique identified sialic acid on the bacterial surface. We isolated lipopolysaccharide from this F. nucleatum strain and performed structural analysis on the O-antigen. Our studies identified a trisaccharide repeating unit of the O-antigen with the following structure: -[→4)-α-Neup5Ac-(2 â†’ 4)-ß-d-Galp-(1 â†’ 3)-α-d-FucpNAc4NAc-(1-]- where Ac indicates 4-N-acetylation of ∼30% FucNAc4N residues. The presence of sialic acid as a constituent of the O-antigen is consistent with recent data identifying de novo sialic acid synthesis in this strain.

Effects of a portion design plate on food group guideline adherence among hospital staff.

Food group guideline adherence is vital to prevent obesity and diabetes. Various studies have demonstrated that environmental variables influence food intake behaviour. In the present study we examined the effect of a portion design plate with food group portion guidelines demarcated by coloured lines (ETE Plate™). A two-group quasi-experimental design was used to measure proportions of carbohydrate, vegetable and protein portions and user experience in a hospital staff lounge setting in Singapore. Lunch was served on the portion design plate before 12.15 hours. For comparison, a normal plate (without markings) was used after 12.15 hours. Changes in proportions of food groups from 2 months before the introduction of the design plate were analysed in a stratified sample at baseline (859 subjects, all on normal plates) to 1, 3 and 6 months after (in all 1016 subjects on the design plate, 968 subjects on the control plate). A total of 151 participants were asked about their experiences and opinions. Between-group comparisons were performed using t tests. Among those served on the portion design plate at 6 months after its introduction, the proportion of vegetables was 4·71 % (P < 0·001) higher and that of carbohydrates 2·83 % (P < 0·001) lower relative to the baseline. No significant change was found for proteins (-1·85 %). Over 6 months, we observed different change patterns between the different food group proportions. While participants were positive about the portion design plate, they did not think it would influence their personal behaviour. A portion design plate might stimulate food group guideline adherence among hospital staff and beyond.

Tannerella forsythia strains display different cell-surface nonulosonic acids: biosynthetic pathway characterization and first insight into biological implications.

Tannerella forsythia is an anaerobic, Gram-negative periodontal pathogen. A unique O-linked oligosaccharide decorates the bacterium's cell surface proteins and was shown to modulate the host immune response. In our study, we investigated the biosynthesis of the nonulosonic acid (NulO) present at the terminal position of this glycan. A bioinformatic analysis of T. forsythia genomes revealed a gene locus for the synthesis of pseudaminic acid (Pse) in the type strain ATCC 43037 while strains FDC 92A2 and UB4 possess a locus for the synthesis of legionaminic acid (Leg) instead. In contrast to the NulO in ATCC 43037, which has been previously identified as a Pse derivative (5-N-acetimidoyl-7-N-glyceroyl-3,5,7,9-tetradeoxy-l-glycero-l-manno-NulO), glycan analysis of strain UB4 performed in this study indicated a 350-Da, possibly N-glycolyl Leg (3,5,7,9-tetradeoxy-d-glycero-d-galacto-NulO) derivative with unknown C5,7 N-acyl moieties. We have expressed, purified and characterized enzymes of both NulO pathways to confirm these genes' functions. Using capillary electrophoresis (CE), CE-mass spectrometry and NMR spectroscopy, our studies revealed that Pse biosynthesis in ATCC 43037 essentially follows the UDP-sugar route described in Helicobacter pylori, while the pathway in strain FDC 92A2 corresponds to Leg biosynthesis in Campylobacter jejuni involving GDP-sugar intermediates. To demonstrate that the NulO biosynthesis enzymes are functional in vivo, we created knockout mutants resulting in glycans lacking the respective NulO. Compared to the wild-type strains, the mutants exhibited significantly reduced biofilm formation on mucin-coated surfaces, suggestive of their involvement in host-pathogen interactions or host survival. This study contributes to understanding possible biological roles of bacterial NulOs.