Glycolanguage of the oral microbiota.

The oral cavity harbors a diverse and dynamic bacterial biofilm community which is pivotal to oral health maintenance and, if turning dysbiotic, can contribute to various diseases. Glycans as unsurpassed carriers of biological information are participating in underlying processes that shape oral health and disease. Bacterial glycoinfrastructure-encompassing compounds as diverse as glycoproteins, lipopolysaccharides (LPSs), cell wall glycopolymers, and exopolysaccharides-is well known to influence bacterial fitness, with direct effects on bacterial physiology, immunogenicity, lifestyle, and interaction and colonization capabilities. Thus, understanding oral bacterias' glycoinfrastructure and encoded glycolanguage is key to elucidating their pathogenicity mechanisms and developing targeted strategies for therapeutic intervention. Driven by their known immunological role, most research in oral glycobiology has been directed onto LPSs, whereas, recently, glycoproteins have been gaining increased interest. This review draws a multifaceted picture of the glycolanguage, with a focus on glycoproteins, manifested in prominent oral bacteria, such as streptococci, Porphyromonas gingivalis, Tannerella forsythia, and Fusobacterium nucleatum. We first define the characteristics of the different glycoconjugate classes and then summarize the current status of knowledge of the structural diversity of glycoconjugates produced by oral bacteria, describe governing biosynthetic pathways, and list biological roles of these energetically costly compounds. Additionally, we highlight emerging research on the unraveling impact of oral glycoinfrastructure on dental caries, periodontitis, and systemic conditions. By integrating current knowledge and identifying knowledge gaps, this review underscores the importance of studying the glycolanguage oral bacteria speak to advance our understanding of oral microbiology and develop novel antimicrobials.

Oral streptococci: modulators of health and disease.

Streptococci are primary colonizers of the oral cavity where they are ubiquitously present and an integral part of the commensal oral biofilm microflora. The role oral streptococci play in the interaction with the host is ambivalent. On the one hand, they function as gatekeepers of homeostasis and are a prerequisite for the maintenance of oral health - they shape the oral microbiota, modulate the immune system to enable bacterial survival, and antagonize pathogenic species. On the other hand, also recognized pathogens, such as oral Streptococcus mutans and Streptococcus sobrinus, which trigger the onset of dental caries belong to the genus Streptococcus. In the context of periodontitis, oral streptococci as excellent initial biofilm formers have an accessory function, enabling late biofilm colonizers to inhabit gingival pockets and cause disease. The pathogenic potential of oral streptococci fully unfolds when their dissemination into the bloodstream occurs; streptococcal infection can cause extra-oral diseases, such as infective endocarditis and hemorrhagic stroke. In this review, the taxonomic diversity of oral streptococci, their role and prevalence in the oral cavity and their contribution to oral health and disease will be discussed, focusing on the virulence factors these species employ for interactions at the host interface.

Molecular modelling and site-directed mutagenesis provide insight into saccharide pyruvylation by the Paenibacillus alvei CsaB enzyme.

Pyruvylation is a biologically versatile but mechanistically unexplored saccharide modification. 4,6-Ketal pyruvylated N-acetylmannosamine within bacterial secondary cell wall polymers serves as a cell wall anchoring epitope for proteins possessing a terminal S-layer homology domain trimer. The pyruvyltransferase CsaB from Paenibacillus alvei served as a model to investigate the structural basis of the pyruvyltransfer reaction by a combination of molecular modelling and site-directed mutagenesis together with an enzyme assay using phosphoenolpyruvate (PEP; donor) and synthetic ß-D-ManNAc-(1 → 4)-α-D-GlcNAc-diphosphoryl-11-phenoxyundecyl (acceptor). CsaB protein structure modelling was done using Phyre2 and I-Tasser based on the partial crystal structure of the Schizosaccharomyces pombe pyruvyltransferase Pvg1p and by AlphaFold. The models informed the construction of twelve CsaB mutants targeted at plausible PEP and acceptor binding sites and KM and kcat values were determined to evaluate the mutants, indicating the importance of a loop region for catalysis. R148, H308 and K328 were found to be critical to PEP binding and insight into acceptor binding was obtained from an analysis of Y14 and F16 mutants, confirming the modelled binding sites and interactions predicted using Molecular Operating Environment. These data lay the basis for future mechanistic studies of saccharide pyruvylation as a novel target for interference with bacterial cell wall assembly.

Multispecies biofilm behavior and host interaction support the association of Tannerella serpentiformis with periodontal health.

The recently identified bacterium Tannerella serpentiformis is the closest phylogenetic relative of Tannerella forsythia, whose presence in oral biofilms is associated with periodontitis. Conversely, T. serpentiformis is considered health-associated. This discrepancy was investigated in a comparative study of the two Tannerella species. The biofilm behavior was analyzed upon their addition and of Porphyromonas gingivalis-each bacterium separately or in combinations-to an in vitro five-species oral model biofilm. Biofilm composition and architecture was analyzed quantitatively using real-time PCR and qualitatively by fluorescence in situ hybridization/confocal laser scanning microscopy, and by scanning electron microscopy. The presence of T. serpentiformis led to a decrease of the total cell number of biofilm bacteria, while P. gingivalis was growth-promoting. This effect was mitigated by T. serpentiformis when added to the biofilm together with P. gingivalis. Notably, T. serpentiformis outcompeted T. forsythia numbers when the two species were simultaneously added to the biofilm compared to biofilms containing T. forsythia alone. Tannerella serpentiformis appeared evenly distributed throughout the multispecies biofilm, while T. forsythia was surface-located. Adhesion and invasion assays revealed that T. serpentiformis was significantly less effective in invading human gingival epithelial cells than T. forsythia. Furthermore, compared to T. forsythia, a higher immunostimulatory potential of human gingival fibroblasts and macrophages was revealed for T. serpentiformis, based on mRNA expression levels of the inflammatory mediators interleukin 6 (IL-6), IL-8, monocyte chemoattractant protein-1 and tumor necrosis factor α, and production of the corresponding proteins. Collectively, these data support the potential of T. serpentiformis to interfere with biological processes relevant to the establishment of periodontitis.

The S-layer homology domains of Paenibacillus alvei surface protein SpaA bind to cell wall polysaccharide through the terminal monosaccharide residue.

Self-assembling (glyco)protein surface layers (S-layers) are ubiquitous prokaryotic cell-surface structures involved in structural maintenance, nutrient diffusion, host adhesion, virulence, and other processes, which makes them appealing targets for therapeutics and biotechnological applications as biosensors or drug delivery systems. However, unlocking this potential requires expanding our understanding of S-layer properties, especially the details of surface-attachment. S-layers of Gram-positive bacteria often are attached through the interaction of S-layer homology (SLH) domain trimers with peptidoglycan-linked secondary cell wall polymers (SCWPs). Cocrystal structures of the SLH domain trimer from the Paenibacillus alvei S-layer protein SpaA (SpaASLH) with synthetic, terminal SCWP disaccharide and trisaccharide analogs, together with isothermal titration calorimetry binding analyses, reveal that while SpaASLH accommodates longer biologically relevant SCWP ligands within both its primary (G2) and secondary (G1) binding sites, the terminal pyruvylated ManNAc moiety serves as the nearly exclusive SCWP anchoring point. Binding is accompanied by displacement of a flexible loop adjacent to the receptor site that enhances the complementarity between protein and ligand, including electrostatic complementarity with the terminal pyruvate moiety. Remarkably, binding of the pyruvylated monosaccharide SCWP fragment alone is sufficient to cause rearrangement of the receptor-binding sites in a manner necessary to accommodate longer SCWP fragments. The observation of multiple conformations in longer oligosaccharides bound to the protein, together with the demonstrated functionality of two of the three SCWP receptor-binding sites, reveals how the SpaASLH-SCWP interaction has evolved to accommodate longer SCWP ligands and alleviate the strain inherent to bacterial S-layer adhesion during growth and division.

Shut-Down of Type IX Protein Secretion Alters the Host Immune Response to Tannerella forsythia and Porphyromonas gingivalis.

Tannerella forsythia and Porphyromonas gingivalis target distinct virulence factors bearing a structurally conserved C-terminal domain (CTD) to the type IX protein secretion system (T9SS). The T9SS comprises an outer membrane translocation complex which works in concert with a signal peptidase for CTD cleavage. Among prominent T9SS cargo linked to periodontal diseases are the TfsA and TfsB components of T. forsythia's cell surface (S-) layer, the bacterium's BspA surface antigen and a set of cysteine proteinases (gingipains) from P. gingivalis. To assess the overall role of the bacterial T9SS in the host response, human macrophages and human gingival fibroblasts were stimulated with T. forsythia and P. gingivalis wild-type bacteria and T9SS signal peptidase-deficient mutants defective in protein secretion, respectively. The immunostimulatory potential of these bacteria was compared by analyzing the mRNA expression levels of the pro-inflammatory mediators IL-6, IL-8, MCP-1 and TNF-α by qPCR and by measuring the production of the corresponding proteins by ELISA. Shot-gun proteomics analysis of T. forsythia and P. gingivalis outer membrane preparations confirmed that several CTD-bearing virulence factors which interact with the human immune system were depleted from the signal peptidase mutants, supportive of effective T9SS shut-down. Three and, more profoundly, 16 hours post stimulation, the T. forsythia T9SS mutant induced significantly less production of cytokines and the chemokine in human cells compared to the corresponding parent strain, while the opposite was observed for the P. gingivalis T9SS mutant. Our data indicate that T9SS shut-down translates into an altered inflammatory response in periodontal pathogens. Thus, the T9SS as a potential novel target for periodontal therapy needs further evaluation.

A Combination of Structural, Genetic, Phenotypic and Enzymatic Analyses Reveals the Importance of a Predicted Fucosyltransferase to Protein O-Glycosylation in the Bacteroidetes.

Diverse members of the Bacteroidetes phylum have general protein O-glycosylation systems that are essential for processes such as host colonization and pathogenesis. Here, we analyzed the function of a putative fucosyltransferase (FucT) family that is widely encoded in Bacteroidetes protein O-glycosylation genetic loci. We studied the FucT orthologs of three Bacteroidetes species-Tannerella forsythia, Bacteroides fragilis, and Pedobacter heparinus. To identify the linkage created by the FucT of B. fragilis, we elucidated the full structure of its nine-sugar O-glycan and found that l-fucose is linked ß1,4 to glucose. Of the two fucose residues in the T. forsythia O-glycan, the fucose linked to the reducing-end galactose was shown by mutational analysis to be l-fucose. Despite the transfer of l-fucose to distinct hexose sugars in the B. fragilis and T. forsythia O-glycans, the FucT orthologs from B. fragilis, T. forsythia, and P. heparinus each cross-complement the B. fragilis ΔBF4306 and T. forsythia ΔTanf_01305 FucT mutants. In vitro enzymatic analyses showed relaxed acceptor specificity of the three enzymes, transferring l-fucose to various pNP-α-hexoses. Further, glycan structural analysis together with fucosidase assays indicated that the T. forsythia FucT links l-fucose α1,6 to galactose. Given the biological importance of fucosylated carbohydrates, these FucTs are promising candidates for synthetic glycobiology.

Assaying Paenibacillus alvei CsaB-Catalysed Ketalpyruvyltransfer to Saccharides by Measurement of Phosphate Release.

Ketalpyruvyltransferases belong to a widespread but little investigated class of enzymes, which utilise phosphoenolpyruvate (PEP) for the pyruvylation of saccharides. Pyruvylated saccharides play pivotal biological roles, ranging from protein binding to virulence. Limiting factors for the characterisation of ketalpyruvyltransferases are the availability of cognate acceptor substrates and a straightforward enzyme assay. We report on a fast ketalpyruvyltransferase assay based on the colorimetric detection of phosphate released during pyruvyltransfer from PEP onto the acceptor via complexation with Malachite Green and molybdate. To optimise the assay for the model 4,6-ketalpyruvyl::ManNAc-transferase CsaB from Paenibacillus alvei, a ß-d-ManNAc-α-d-GlcNAc-diphosphoryl-11-phenoxyundecyl acceptor mimicking an intermediate of the bacterium's cell wall glycopolymer biosynthesis pathway, upon which CsaB is naturally active, was produced chemo-enzymatically and used together with recombinant CsaB. Optimal assay conditions were 5 min reaction time at 37 °C and pH 7.5, followed by colour development for 1 h at 37 °C and measurement of absorbance at 620 nm. The structure of the generated pyruvylated product was confirmed by NMR spectroscopy. Using the established assay, the first kinetic constants of a 4,6-ketalpyuvyl::ManNAc-transferase could be determined; upon variation of the acceptor and PEP concentrations, a KM, PEP of 19.50 ± 3.50 µM and kcat, PEP of 0.21 ± 0.01 s-1 as well as a KM, Acceptor of 258 ± 38 µM and a kcat, Acceptor of 0.15 ± 0.01 s-1 were revealed. P. alvei CsaB was inactive on synthetic pNP-ß-d-ManNAc and ß-d-ManNAc-ß-d-GlcNAc-1-OMe, supporting the necessity of a complex acceptor substrate.

Mutanobactin D from the Human Microbiome: Total Synthesis, Configurational Assignment, and Biological Evaluation.

Mutanobactin D is a non-ribosomal, cyclic peptide isolated from Streptococcus mutans and shows activity reducing yeast-to-hyphae transition as well as biofilm formation of the pathogenic yeast Candida albicans. We report the first total synthesis of this natural product, which relies on enantioselective, zinc-mediated 1,3-dipolar cycloaddition and a sequence of cascading reactions, providing the key lipidated γ-amino acid found in mutanobactin D. The synthesis enables configurational assignment, determination of the dominant solution-state structure, and studies to assess the stability of the lipopeptide substructure found in the natural product. The information stored in the fingerprint region of the IR spectra in combination with quantum chemical calculations proved key to distinguishing between epimers of the α-substituted ß-keto amide. Synthetic mutanobactin D drives discovery and analysis of its effect on growth of other members of the human oral consortium. Our results showcase how total synthesis is central for elucidating the complex network of interspecies communications of human colonizers.

Peptidoglycan Salvage Enables the Periodontal Pathogen Tannerella forsythia to Survive within the Oral Microbial Community.

Tannerella forsythia is an anaerobic, fusiform Gram-negative oral pathogen strongly associated with periodontitis, a multibacterial inflammatory disease that leads to the destruction of the teeth-supporting tissue, ultimately causing tooth loss. To survive in the oral habitat, T. forsythia depends on cohabiting bacteria for the provision of nutrients. For axenic growth under laboratory conditions, it specifically relies on the external supply of N-acetylmuramic acid (MurNAc), which is an essential constituent of the peptidoglycan (PGN) of bacterial cell walls. T. forsythia comprises a typical Gram-negative PGN; however, as evidenced by genome sequence analysis, the organism lacks common enzymes required for the de novo synthesis of precursors of PGN, which rationalizes its MurNAc auxotrophy. Only recently insights were obtained into how T. forsythia gains access to MurNAc in its oral habitat, enabling synthesis of the own PGN cell wall. This report summarizes T. forsythia's strategies to survive in the oral habitat by means of PGN salvage pathways, including recovery of exogenous MurNAc and PGN-derived fragments but also polymeric PGN, which are all derived from cohabiting bacteria either via cell wall turnover or decay of cells. Salvage of polymeric PGN presumably requires the removal of peptides from PGN by an unknown amidase, concomitantly with the translocation of the polymer across the outer membrane. Two recently identified exo-lytic N-acetylmuramidases (Tf_NamZ1 and Tf_NamZ2) specifically cleave the peptide-free, exogenous (nutrition source) PGN in the periplasm and release the MurNAc and disaccharide substrates for the transporters Tf_MurT and Tf_AmpG, respectively, whereas the peptide-containing, endogenous (the self-cell wall) PGN stays unattached. This review also outlines how T. forsythia synthesises the PGN precursors UDP-MurNAc and UDP-N-acetylglucosamine (UDP-GlcNAc), involving homologs of the Pseudomonas sp. recycling enzymes AmgK/MurU and a monofunctional uridylyl transferase (named Tf_GlmU*), respectively.

LytR-CpsA-Psr Glycopolymer Transferases: Essential Bricks in Gram-Positive Bacterial Cell Wall Assembly.

The cell walls of Gram-positive bacteria contain a variety of glycopolymers (CWGPs), a significant proportion of which are covalently linked to the peptidoglycan (PGN) scaffolding structure. Prominent CWGPs include wall teichoic acids of Staphylococcus aureus, streptococcal capsules, mycobacterial arabinogalactan, and rhamnose-containing polysaccharides of lactic acid bacteria. CWGPs serve important roles in bacterial cellular functions, morphology, and virulence. Despite evident differences in composition, structure and underlaying biosynthesis pathways, the final ligation step of CWGPs to the PGN backbone involves a conserved class of enzymes-the LytR-CpsA-Psr (LCP) transferases. Typically, the enzymes are present in multiple copies displaying partly functional redundancy and/or preference for a distinct CWGP type. LCP enzymes require a lipid-phosphate-linked glycan precursor substrate and catalyse, with a certain degree of promiscuity, CWGP transfer to PGN of different maturation stages, according to in vitro evidence. The prototype attachment mode is that to the C6-OH of N-acetylmuramic acid residues via installation of a phosphodiester bond. In some cases, attachment proceeds to N-acetylglucosamine residues of PGN-in the case of the Streptococcus agalactiae capsule, even without involvement of a phosphate bond. A novel aspect of LCP enzymes concerns a predicted role in protein glycosylation in Actinomyces oris. Available crystal structures provide further insight into the catalytic mechanism of this biologically important class of enzymes, which are gaining attention as new targets for antibacterial drug discovery to counteract the emergence of multidrug resistant bacteria.

Utilization of different MurNAc sources by the oral pathogen Tannerella forsythia and role of the inner membrane transporter AmpG.

BACKGROUND: The Gram-negative oral pathogen Tannerella forsythia strictly depends on the external supply of the essential bacterial cell wall sugar N-acetylmuramic acid (MurNAc) for survival because of the lack of the common MurNAc biosynthesis enzymes MurA/MurB. The bacterium thrives in a polymicrobial biofilm consortium and, thus, it is plausible that it procures MurNAc from MurNAc-containing peptidoglycan (PGN) fragments (muropeptides) released from cohabiting bacteria during natural PGN turnover or cell death. There is indirect evidence that in T. forsythia, an AmpG-like permease (Tanf_08365) is involved in cytoplasmic muropeptide uptake. In E. coli, AmpG is specific for the import of N-acetylglucosamine (GlcNAc)-anhydroMurNAc(-peptides) which are common PGN turnover products, with the disaccharide portion as a minimal requirement. Currently, it is unclear which natural, complex MurNAc sources T. forsythia can utilize and which role AmpG plays therein. RESULTS: We performed a screen of various putative MurNAc sources for T. forsythia mimicking the situation in the natural habitat and compared bacterial growth and cell morphology of the wild-type and a mutant lacking AmpG (T. forsythia ΔampG). We showed that supernatants of the oral biofilm bacteria Porphyromonas gingivalis and Fusobacterium nucleatum, and of E. coli ΔampG, as well as isolated PGN and defined PGN fragments obtained after enzymatic digestion, namely GlcNAc-anhydroMurNAc(-peptides) and GlcNAc-MurNAc(-peptides), could sustain growth of T. forsythia wild-type, while T. forsythia ΔampG suffered from growth inhibition. In supernatants of T. forsythia ΔampG, the presence of GlcNAc-anhMurNAc and, unexpectedly, also GlcNAc-MurNAc was revealed by tandem mass spectrometry analysis, indicating that both disaccharides are substrates of AmpG. The importance of AmpG in the utilization of PGN fragments as MurNAc source was substantiated by a significant ampG upregulation in T. forsythia cells cultivated with PGN, as determined by quantitative real-time PCR. Further, our results indicate that PGN-degrading amidase, lytic transglycosylase and muramidase activities in a T. forsythia cell extract are involved in PGN scavenging. CONCLUSION: T. forsythia metabolizes intact PGN as well as muropeptides released from various bacteria and the bacterium's inner membrane transporter AmpG is essential for growth on these MurNAc sources, and, contrary to the situation in E. coli, imports both, GlcNAc-anhMurNAc and GlcNAc-MurNAc fragments.

The Intriguing Interaction of Escherichia coli with the Host Environment and Innovative Strategies To Interfere with Colonization: a Summary of the 2019 E. coli and the Mucosal Immune System Meeting.

The third E. coli and the Mucosal Immune System (ECMIS) meeting was held at Ghent University in Belgium from 2 to 5 June 2019. It brought together an international group of scientists interested in mechanisms of colonization, host response, and vaccine development. ECMIS distinguishes itself from related meetings on these enteropathogens by providing a greater emphasis on animal health and disease and covering a broad range of pathotypes, including enterohemorrhagic, enteropathogenic, enterotoxigenic, enteroaggregative, and extraintestinal pathogenic Escherichia coli As it is well established that the genus Shigella represents a subspecies of E. coli, these organisms along with related enteroinvasive E. coli are also included. In addition, Tannerella forsythia, a periodontal pathogen, was presented as an example of a pathogen which uses its surface glycans for mucosal interaction. This review summarizes several highlights from the 2019 meeting and major advances to our understanding of the biology of these pathogens and their impact on the host.

Response of Human Mesenchymal Stromal Cells from Periodontal Tissue to LPS Depends on the Purity but Not on the LPS Source.

Human periodontal ligament stromal cells (hPDLSCs) and gingival mesenchymal stromal cells (hGMSCs) are resident mesenchymal stromal cells (MSCs) of the periodontal tissue. The lipopolysaccharide (LPS) from Porphyromonas gingivalis is structurally distinct from that of other Gram-negative bacteria, and earlier studies linked this structural difference to a distinct virulence activity and the ability to activate toll-like receptor 2 (TLR-2), besides TLR-4 as commonly occurring upon LPS challenge. Later studies, in contrast, argue that TLR-2 activation by P. gingivalis LPS is due to lipoprotein contamination. In the present study, we aimed to define the influence of structure versus purity of P. gingivalis LPS on the immune response of hPDLSCs and hGMSCs. Cells were stimulated with commercially available "standard" P. gingivalis LPS, "ultrapure" P. gingivalis LPS, or "ultrapure" Escherichia coli LPS, and the expression of interleukin- (IL-) 8, IL-6, monocyte chemoattractant protein- (MCP-) 1, TLR-2, and TLR-4 was evaluated. The contribution of TLR-4 to the LPS-induced response was assessed using the specific TLR-4 inhibitor TAK-242. "Standard" P. gingivalis LPS induced significantly higher IL-8, IL-6, and MCP-1 production compared to the "ultrapure" LPS preparations, with no significant difference detectable for "ultrapure" LPS from P. gingivalis and E. coli. By using TAK-242, the response of hPDLSCs and hGMSCs to "ultrapure" LPS preparations was effectively inhibited to the levels comparable to those of nonstimulated controls. In contrast, high levels of response to "standard" LPS were observed, even in the presence of TAK-242. Our data show that the response of MSCs from periodontal tissue to LPS depends more on the purity of the LPS preparation than on the LPS source. Even a small amount of contaminating lipoproteins can drastically enhance the hPDLSCs' and hGMSCs; responsiveness to P. gingivalis LPS, which might also contribute to the progression of periodontal disease.

Comparative genome characterization of the periodontal pathogen Tannerella forsythia.

BACKGROUND: Tannerella forsythia is a bacterial pathogen implicated in periodontal disease. Numerous virulence-associated T. forsythia genes have been described, however, it is necessary to expand the knowledge on T. forsythia's genome structure and genetic repertoire to further elucidate its role within pathogenesis. Tannerella sp. BU063, a putative periodontal health-associated sister taxon and closest known relative to T. forsythia is available for comparative analyses. In the past, strain confusion involving the T. forsythia reference type strain ATCC 43037 led to discrepancies between results obtained from in silico analyses and wet-lab experimentation. RESULTS: We generated a substantially improved genome assembly of T. forsythia ATCC 43037 covering 99% of the genome in three sequences. Using annotated genomes of ten Tannerella strains we established a soft core genome encompassing 2108 genes, based on orthologs present in > = 80% of the strains analysed. We used a set of known and hypothetical virulence factors for comparisons in pathogenic strains and the putative periodontal health-associated isolate Tannerella sp. BU063 to identify candidate genes promoting T. forsythia's pathogenesis. Searching for pathogenicity islands we detected 38 candidate regions in the T. forsythia genome. Only four of these regions corresponded to previously described pathogenicity islands. While the general protein O-glycosylation gene cluster of T. forsythia ATCC 43037 has been described previously, genes required for the initiation of glycan synthesis are yet to be discovered. We found six putative glycosylation loci which were only partially conserved in other bacteria. Lastly, we performed a comparative analysis of translational bias in T. forsythia and Tannerella sp. BU063 and detected highly biased genes. CONCLUSIONS: We provide resources and important information on the genomes of Tannerella strains. Comparative analyses enabled us to assess the suitability of T. forsythia virulence factors as therapeutic targets and to suggest novel putative virulence factors. Further, we report on gene loci that should be addressed in the context of elucidating T. forsythia's protein O-glycosylation pathway. In summary, our work paves the way for further molecular dissection of T. forsythia biology in general and virulence of this species in particular.

Pyruvate Substitutions on Glycoconjugates.

Glycoconjugates are the most diverse biomolecules of life. Mostly located at the cell surface, they translate into cell-specific "barcodes" and offer a vast repertoire of functions, including support of cellular physiology, lifestyle, and pathogenicity. Functions can be fine-tuned by non-carbohydrate modifications on the constituting monosaccharides. Among these modifications is pyruvylation, which is present either in enol or ketal form. The most commonly best-understood example of pyruvylation is enol-pyruvylation of N-acetylglucosamine, which occurs at an early stage in the biosynthesis of the bacterial cell wall component peptidoglycan. Ketal-pyruvylation, in contrast, is present in diverse classes of glycoconjugates, from bacteria to algae to yeast-but not in humans. Mild purification strategies preventing the loss of the acid-labile ketal-pyruvyl group have led to a collection of elucidated pyruvylated glycan structures. However, knowledge of involved pyruvyltransferases creating a ring structure on various monosaccharides is scarce, mainly due to the lack of knowledge of fingerprint motifs of these enzymes and the unavailability of genome sequences of the organisms undergoing pyruvylation. This review compiles the current information on the widespread but under-investigated ketal-pyruvylation of monosaccharides, starting with different classes of pyruvylated glycoconjugates and associated functions, leading to pyruvyltransferases, their specificity and sequence space, and insight into pyruvate analytics.

Peptidoglycan-type analysis of the N-acetylmuramic acid auxotrophic oral pathogen Tannerella forsythia and reclassification of the peptidoglycan-type of Porphyromonas gingivalis.

BACKGROUND: Tannerella forsythia is a Gram-negative oral pathogen. Together with Porphyromonas gingivalis and Treponema denticola it constitutes the "red complex" of bacteria, which is crucially associated with periodontitis, an inflammatory disease of the tooth supporting tissues that poses a health burden worldwide. Due to the absence of common peptidoglycan biosynthesis genes, the unique bacterial cell wall sugar N-acetylmuramic acid (MurNAc) is an essential growth factor of T. forsythia to build up its peptidoglycan cell wall. Peptidoglycan is typically composed of a glycan backbone of alternating N-acetylglucosamine (GlcNAc) and MurNAc residues that terminates with anhydroMurNAc (anhMurNAc), and short peptides via which the sugar backbones are cross-linked to build up a bag-shaped network. RESULTS: We investigated T. forsythia's peptidoglycan structure, which is an essential step towards anti-infective strategies against this pathogen. A new sensitive radioassay was developed which verified the presence of MurNAc and anhMurNAc in the cell wall of the bacterium. Upon digest of isolated peptidoglycan with endo-N-acetylmuramidase, exo-N-acetylglucosaminidase and muramyl-L-alanine amidase, respectively, peptidoglycan fragments were obtained. HPLC and mass spectrometry (MS) analyses revealed the presence of GlcNAc-MurNAc-peptides and the cross-linked dimer with retention-times and masses, respectively, equalling those of control digests of Escherichia coli and P. gingivalis peptidoglycan. Data were confirmed by tandem mass spectrometry (MS2) analysis, revealing the GlcNAc-MurNAc-tetra-tetra-MurNAc-GlcNAc dimer to contain the sequence of the amino acids alanine, glutamic acid, diaminopimelic acid (DAP) and alanine, as well as a direct cross-link between DAP on the third and alanine on the fourth position of the two opposite stem peptides. The stereochemistry of DAP was determined by reversed-phase HPLC after dabsylation of hydrolysed peptidoglycan to be of the meso-type. CONCLUSION: T. forsythia peptidoglycan is of the A1γ-type like that of E. coli. Additionally, the classification of P. gingivalis peptidoglycan as A3γ needs to be revised to A1γ, due to the presence of meso-DAP instead of LL-DAP, as reported previously.

Nonulosonic acids contribute to the pathogenicity of the oral bacterium Tannerella forsythia.

Periodontitis is a polymicrobial, biofilm-caused, inflammatory disease affecting the tooth-supporting tissues. It is not only the leading cause of tooth loss worldwide, but can also impact systemic health. The development of effective treatment strategies is hampered by the complicated disease pathogenesis which is best described by a polymicrobial synergy and dysbiosis model. This model classifies the Gram-negative anaerobe Tannerella forsythia as a periodontal pathogen, making it a prime candidate for interference with the disease. Tannerella forsythia employs a protein O-glycosylation system that enables high-density display of nonulosonic acids via the bacterium's two-dimensional crystalline cell surface layer. Nonulosonic acids are sialic acid-like sugars which are well known for their pivotal biological roles. This review summarizes the current knowledge of T. forsythia's unique cell envelope with a focus on composition, biosynthesis and functional implications of the cell surface O-glycan. We have obtained evidence that glycobiology affects the bacterium's immunogenicity and capability to establish itself in the polymicrobial oral biofilm. Analysis of the genomes of different T. forsythia isolates revealed that complex protein O-glycosylation involving nonulosonic acids is a hallmark of pathogenic T. forsythia strains and, thus, constitutes a valuable target for the design of novel anti-infective strategies to combat periodontitis.

Assaying Fucosidase Activity.

The characterization of a recombinant glycosidase can be done with commercially available substrates, which enable testing of enzyme functionality and determination of linkage specificity. Colorimetric assays with p-nitrophenyl substrates provide a relatively simple and fast way of screening conditions which could affect enzyme activity (buffer, pH, ion dependence, temperature). These substrates are useful for the determination of activity optima and the characterization of basic activity parameters. However, testing for linkage specificity should be performed on more complex sugars presenting a range of different glycosidic bonds and might need more sophisticated methods of analysis. This protocol provides comprehensive instructions on how to perform an initial characterization of your glycosidase using a recombinant α-L-fucosidase as an example.

Salutogenic Affordances and Sustainability: Multiple Benefits With Edible Forest Gardens in Urban Green Spaces.

With increased urbanization, ecological challenges such as climate change and loss of biodiversity, and stress-related disorders globally posing a major threat to public health and wellbeing, the development of efficient multiple-use strategies for urban green spaces and infrastructures is of great importance. In addition to benefits such as climate and water regulation, food production, and biodiversity conservation, green spaces and features have been associated with various health and wellbeing outcomes from a psychological perspective. Research suggests links between exposure to green environmental qualities and restoration from psycho-physiological stress and attention fatigue, promotion of physical activity, increased neighborhood satisfaction and even reduced mortality. Especially strong associations have been observed in urban and socio-economically challenged areas. Usually such salutogenic, i.e., health-promoting, effects are explained through theories related to the notion of biophilia, i.e., the idea that humans share innate tendencies to attend to natural environments and features that have been beneficial during evolution. This paper assumes an ecological approach to perception and behavior to be fruitful in order to analyze the salutogenic potential of environments such as urban green spaces and to step beyond the "green vs. gray" dichotomy that has been prevalent through much of the research on health-promoting environments. Through an analysis of environmental affordances for certain perceived qualities such an approach is explored through a proposed concept for urban green space use and management, the edible forest garden. Such gardens, based on agroecological principles, have emerged as one of the most promising models regarding ecologically sustainable food production. In addition to potential contributions of importance for urban sustainability and biodiversity, we argue that the inclusion of edible forest gardens in urban green spaces - today globally dominated by lawns - also potentially could reinforce several affordances of salutogenic importance, both in terms of, e.g., social cohesion but also in regard to restoration from psycho-physiological stress and attention fatigue. Increased opportunities for contact with nature and processes of food production may also reinforce pro-environmental behaviors in the population and thus also affect long-term sustainability.