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, bacterium, pseudaminic and legionaminic acid

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
5,0,0,2	Ac
5,0,0	aD6dxylHexpN-4-ulo	95.5	53.7	72.5	?	71.1	12.5
5,0	P
5	P
	xXnucG


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
5,0,0,2	Ac
5,0,0	aD6dxylHexpN-4-ulo	5.46	4.09	3.83	-	4.11	1.21
5,0	P
5	P
	xXnucG


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
5,0,0,2	Ac
5,0,0	aD6dxylHexpN-4-ulo	95.5/5.46	53.7/4.09	72.5/3.83		71.1/4.11	12.5/1.21
5,0	P
5	P
	xXnucG

The spectrum also has 1 signal at unknown position (not plotted).

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

CC(=O)N[C@H]1[C@@H](OP(=O)(O)OP(=O)(O)OC[C@H]2O[C@@H](n3cnc4c(=O)[nH]c(N)nc43)[C@H](O)[C@@H]2O)O[C@H](C)C(=O)[C@@H]1O

628.381 g/mol (C₁₈H₂₆N₆O₁₅P₂)