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.

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.

Cytoplasmic glycoengineering enables biosynthesis of nanoscale glycoprotein assemblies.

Glycosylation of proteins profoundly impacts their physical and biological properties. Yet our ability to engineer novel glycoprotein structures remains limited. Established bacterial glycoengineering platforms require secretion of the acceptor protein to the periplasmic space and preassembly of the oligosaccharide substrate as a lipid-linked precursor, limiting access to protein and glycan substrates respectively. Here, we circumvent these bottlenecks by developing a facile glycoengineering platform that operates in the bacterial cytoplasm. The Glycoli platform leverages a recently discovered site-specific polypeptide glycosyltransferase together with variable glycosyltransferase modules to synthesize defined glycans, of bacterial or mammalian origin, directly onto recombinant proteins in the E. coli cytoplasm. We exploit the cytoplasmic localization of this glycoengineering platform to generate a variety of multivalent glycostructures, including self-assembling nanomaterials bearing hundreds of copies of the glycan epitope. This work establishes cytoplasmic glycoengineering as a powerful platform for producing glycoprotein structures with diverse future biomedical applications.

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.

A General Protein O-Glycosylation Gene Cluster Encodes the Species-Specific Glycan of the Oral Pathogen Tannerella forsythia: O-Glycan Biosynthesis and Immunological Implications.

The cell surface of the oral pathogen Tannerella forsythia is heavily glycosylated with a unique, complex decasaccharide that is O-glycosidically linked to the bacterium's abundant surface (S-) layer, as well as other proteins. The S-layer glycoproteins are virulence factors of T. forsythia and there is evidence that protein O-glycosylation underpins the bacterium's pathogenicity. To elucidate the protein O-glycosylation pathway, genes suspected of encoding pathway components were first identified in the genome sequence of the ATCC 43037 type strain, revealing a 27-kb gene cluster that was shown to be polycistronic. Using a gene deletion approach targeted at predicted glycosyltransferases (Gtfs) and methyltransferases encoded in this gene cluster, in combination with mass spectrometry of the protein-released O-glycans, we show that the gene cluster encodes the species-specific part of the T. forsythia ATCC 43037 decasaccharide and that this is assembled step-wise on a pentasaccharide core. The core was previously proposed to be conserved within the Bacteroidetes phylum, to which T. forsythia is affiliated, and its biosynthesis is encoded elsewhere on the bacterial genome. Next, to assess the prevalence of protein O-glycosylation among Tannerella sp., the publicly available genome sequences of six T. forsythia strains were compared, revealing gene clusters of similar size and organization as found in the ATCC 43037 type strain. The corresponding region in the genome of a periodontal health-associated Tannerella isolate showed a different gene composition lacking most of the genes commonly found in the pathogenic strains. Finally, we investigated whether differential cell surface glycosylation impacts T. forsythia's overall immunogenicity. Release of proinflammatory cytokines by dendritic cells (DCs) upon stimulation with defined Gtf-deficient mutants of the type strain was measured and their T cell-priming potential post-stimulation was explored. This revealed that the O-glycan is pivotal to modulating DC effector functions, with the T. forsythia-specific glycan portion suppressing and the pentasaccharide core activating a Th17 response. We conclude that complex protein O-glycosylation is a hallmark of pathogenic T. forsythia strains and propose it as a valuable target for the design of novel antimicrobials against periodontitis.

N-Acetylmuramic Acid (MurNAc) Auxotrophy of the Oral Pathogen Tannerella forsythia: Characterization of a MurNAc Kinase and Analysis of Its Role in Cell Wall Metabolism.

Tannerella forsythia is an anaerobic, Gram-negative oral pathogen that thrives in multispecies gingival biofilms associated with periodontitis. The bacterium is auxotrophic for the commonly essential bacterial cell wall sugar N-acetylmuramic acid (MurNAc) and, thus, strictly depends on an exogenous supply of MurNAc for growth and maintenance of cell morphology. A MurNAc transporter (Tf_MurT; Tanf_08375) and an ortholog of the Escherichia coli etherase MurQ (Tf_MurQ; Tanf_08385) converting MurNAc-6-phosphate to GlcNAc-6-phosphate were recently described for T. forsythia. In between the respective genes on the T. forsythia genome, a putative kinase gene is located. In this study, the putative kinase (Tf_MurK; Tanf_08380) was produced as a recombinant protein and biochemically characterized. Kinetic studies revealed Tf_MurK to be a 6-kinase with stringent substrate specificity for MurNAc exhibiting a 6 × 104-fold higher catalytic efficiency (kcat/Km ) for MurNAc than for N-acetylglucosamine (GlcNAc) with kcat values of 10.5 s-1 and 0.1 s-1 and Km values of 200 µM and 116 mM, respectively. The enzyme kinetic data suggest that Tf_MurK is subject to substrate inhibition (Ki[S] = 4.2 mM). To assess the role of Tf_MurK in the cell wall metabolism of T. forsythia, a kinase deletion mutant (ΔTf_murK::erm) was constructed. This mutant accumulated MurNAc intracellularly in the exponential phase, indicating the capability to take up MurNAc, but inability to catabolize MurNAc. In the stationary phase, the MurNAc level was reduced in the mutant, while the level of the peptidoglycan precursor UDP-MurNAc-pentapeptide was highly elevated. Further, according to scanning electron microscopy evidence, the ΔTf_murK::erm mutant was more tolerant toward low MurNAc concentration in the medium (below 0.5 µg/ml) before transition from healthy, rod-shaped to fusiform cells occurred, while the parent strain required > 1 µg/ml MurNAc for optimal growth. These data reveal that T. forsythia readily catabolizes exogenous MurNAc but simultaneously channels a proportion of the sugar into peptidoglycan biosynthesis. Deletion of Tf_murK blocks MurNAc catabolism and allows the direction of MurNAc solely to peptidoglycan biosynthesis, resulting in a growth advantage in MurNAc-depleted medium. This work increases our understanding of the T. forsythia cell wall metabolism and may pave new routes for lead finding in the treatment of periodontitis.

A pseudaminic acid or a legionaminic acid derivative transferase is strain-specifically implicated in the general protein O-glycosylation system of the periodontal pathogen Tannerella forsythia.

The occurrence of nonulosonic acids in bacteria is wide-spread and linked to pathogenicity. However, the knowledge of cognate nonulosonic acid transferases is scarce. In the periodontopathogen Tannerella forsythia, several proposed virulence factors carry strain-specifically either a pseudaminic or a legionaminic acid derivative as terminal sugar on an otherwise structurally identical, protein-bound oligosaccharide. This study aims to shed light on the transfer of either nonulosonic acid derivative on a proximal N-acetylmannosaminuronic acid residue within the O-glycan structure, exemplified with the bacterium's abundant S-layer glycoproteins. Bioinformatic analyses provided the candidate genes Tanf_01245 (strain ATCC 43037) and TFUB4_00887 (strain UB4), encoding a putative pseudaminic and a legionaminic acid derivative transferase, respectively. These transferases have identical C-termini and contain motifs typical of glycosyltransferases (DXD) and bacterial sialyltransferases (D/E-D/E-G and HP). They share homology to type B glycosyltransferases and TagB, an enzyme catalyzing glycerol transfer to an N-acetylmannosamine residue in teichoic acid biosynthesis. Analysis of a cellular pool of nucleotide-activated sugars confirmed the presence of the CMP-activated nonulosonic acid derivatives, which are most likely serving as substrates for the corresponding transferase. Single gene knock-out mutants targeted at either transferase were analyzed for S-layer O-glycan composition by ESI-MS, confirming the loss of the nonulosonic acid derivative. Cross-complementation of the mutants with the nonnative nonulosonic acid transferase was not successful indicating high stringency of the enzymes. This study identified plausible candidates for a pseudaminic and a legionaminic acid derivative transferase; these may serve as valuable tools for engineering of novel sialoglycoconjugates.

Unexpected T-cell recognition of an altered peptide ligand is driven by reversed thermodynamics.

The molecular basis underlying T-cell recognition of MHC molecules presenting altered peptide ligands is still not well-established. A hierarchy of T-cell activation by MHC class I-restricted altered peptide ligands has been defined using the T-cell receptor P14 specific for H-2D(b) in complex with the immunodominant lymphocytic choriomeningitis virus peptide gp33 (KAVYNFATM). While substitution of tyrosine to phenylalanine (Y4F) or serine (Y4S) abolished recognition by P14, the TCR unexpectedly recognized H-2D(b) in complex with the alanine-substituted semiagonist Y4A, which displayed the most significant structural modification. The observed functional hierarchy gp33 > Y4A > Y4S = Y4F was neither due to higher stabilization capacity nor to differences in structural conformation. However, thermodynamic analysis demonstrated that while recognition of the full agonist H-2D(b) /gp33 was strictly enthalpy driven, recognition of the weak agonist H-2D(b) /Y4A was instead entropy driven with a large reduction in the favorable enthalpy term. The fourfold larger negative heat capacity derived for the interaction of P14 with H-2D(b) /gp33 compared with H-2D(b) /Y4A can possibly be explained by higher water entrapment at the TCR/MHC interface, which is also consistent with the measured opposite entropy contributions for the interactions of P14 with both MHCs. In conclusion, this study demonstrates that P14 makes use of different strategies to adapt to structural modifications in the MHC/peptide complex.