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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 β-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-β-d-ManNAc and β-d-ManNAc-β-d-GlcNAc-1-OMe, supporting the necessity of a complex acceptor substrate.

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 β1,4 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.

Glycobiology is important for the periodontal pathogen Tannerella forsythia, affecting the bacterium’s cellular integrity, its life-style, and virulence potential. The bacterium possesses a unique Gram-negative cell envelope with a glycosylated surface (S-) layer as outermost decoration that is proposed to be anchored via a rough lipopolysaccharide. The S-layer glycan has the structure 4‑MeO-b-ManpNAcCONH2-(1→3)-[Pse5Am7Gc-(2→4)-]-b-ManpNAcA-(1→4)-[4-MeO-a-Galp-(1→2)-]-a-Fucp-(1→4)-[-a-Xylp-(1→3)-]-b-GlcpA-(1→3)-[-b-Digp-(1→2)-]-a-Galp and is linked to distinct serine and threonine residues within the D(S/T)(A/I/L/M/T/V) amino acid motif. Also several other Tannerella proteins are modified with the S‑layer oligosaccharide, indicating the presence of a general O‑glycosylation system. Protein O‑glycosylation impacts the life-style of T. forsythia since truncated S-layer glycans present in a defined mutant favor biofilm formation. While the S‑layer has also been shown to be a virulence factor and to delay the bacterium's recognition by the innate immune system of the host, the contribution of glycosylation to modulating host immunity is currently unraveling. Recently, it was shown that Tannerella surface glycosylation has a role in restraining the Th17-mediated neutrophil infiltration in the gingival tissues. Related to its asaccharolytic physiology, T. forsythia expresses a robust enzymatic repertoire, including several glycosidases, such as sialidases, which are linked to specific growth requirements and are involved in triggering host tissue destruction. This review compiles the current knowledge on the glycobiology of T. forsythia.