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.

Campylobacter jejuni, Helicobacter, Biofilm, biosynthesis pathway, pseudaminic and legionaminic acid, bacterium

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6	C7	C8	C9
5,0,5	Ac
5,0,7	Ac
5,0	?XPse?	?	?	37.1	66.1	50.0	73.9	55.0	69.9	18.4
5	P
	xXnucC


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6	H7	H8	H9
5,0,5	Ac
5,0,7	Ac
5,0	?XPse?	-	-	1.60-2.21	4.23	4.28	4.29	4.02	4.11	1.19
5	P
	xXnucC


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6	C7/H7	C8/H8	C9/H9
5,0,5	Ac
5,0,7	Ac
5,0	?XPse?			37.1/1.60-2.21	66.1/4.23	50.0/4.28	73.9/4.29	55.0/4.02	69.9/4.11	18.4/1.19
5	P
	xXnucC

The spectrum also has 2 signals at unknown positions (not plotted).

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

CC(=O)N[C@@H]([C@H](C)O)[C@@H]1OC(OP(=O)(O)OC[C@H]2O[C@@H](n3ccc(N)nc3=O)[C@H](O)[C@@H]2O)(C(=O)O)C[C@H](O)[C@@H]1NC(C)=O

639.508 g/mol (C₂₂H₃₄N₅O₁₅P)

Campylobacter jejuni has systems for N- and O-linked protein glycosylation. Although biochemical evidence demonstrated that a pseC mutant in the O-linked pathway accumulated the product of pglF in the N-linked pathway, analyses of transformation frequencies and glycosylation statuses of N-glycosylated proteins indicated a partial suppression of pglF by pseC

biosynthesis, Bacterial Proteins, Campylobacter jejuni, glycosylation

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

CC(=O)N[C@@H]([C@H](C)O)[C@@H]1OC(OP(=O)(O)OC[C@H]2O[C@@H](n3ccc(N)nc3=O)[C@H](O)[C@@H]2O)(C(=O)O)C[C@H](O)[C@@H]1NC(C)=O

639.508 g/mol (C₂₂H₃₄N₅O₁₅P)

Glycomics which is the study of saccharides and genes responsible for their formation requires the continuous development of rapid and sensitive methods for the identification of glycan structures. It involves glycoanalysis which relies upon the development of methods for determining the structure and interactions of carbohydrates. For the application of functional glycomics to microbial virulence, carbohydrates and their associated metabolic and carbohydrate processing enzymes and respective genes can be identified and exploited as targets for drug discovery, glyco-engineering, vaccine design, and detection and diagnosis of diseases. Glycomics also encompasses the detailed understanding of carbohydrate-protein interactions and this knowledge can be applied to research efforts focused toward the development of vaccines and immunological therapies to alleviate infectious diseases.

NMR, polysaccharides, structural analysis, molecular modeling, HR-MAS, Glycomics, glycans, glycoanalysis, protein–carbohydrate interactions

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

CC(=O)N[C@@H]([C@H](C)O)[C@@H]1OC(OP(=O)(O)OC[C@H]2O[C@@H](n3ccc(N)nc3=O)[C@H](O)[C@@H]2O)(C(=O)O)C[C@H](O)[C@@H]1NC(C)=O

639.508 g/mol (C₂₂H₃₄N₅O₁₅P)