Tannerella forsythiastrains display different cell-surface nonulosonic acids: biosynthetic pathway characterization and first insight into biological implications

Abstract

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.

Immune response profiling of primary monocytes and oral keratinocytes to different Tannerella forsythia strains and their cell surface mutants

“…A prominent characteristic of T. forsythia is its elaborate cell surface structure consisting of the two‐dimensional crystalline S‐layer with attached, highly complex O ‐glycans . The two T. forsythia wild‐type strains ATCC 43037 and UB4 differ in the terminal sugar residue of an otherwise identical dekasaccharide, which is present as a modified Pse in T. forsythia ATCC 43037 and a modified Leg in T. forsythia UB4 . Including in this study mutants with selected cell surface defects, we investigated to which extent the molecular mimics of sialic acid—Pse and Leg—as well as the T. forsythia S‐layer and attached glycan influence the host immune response.…”

“…26 The two T. forsythia wild-type strains ATCC 43037 and UB4 differ in the terminal sugar residue of an otherwise identical dekasaccharide, which is present as a modified Pse in T. forsythia ATCC 43037 and a modified Leg in T. forsythia UB4. 29 Including in this study mutants with selected cell surface defects, we investigated to which extent the molecular mimics of sialic acid-Pse forsythia in macrophages and human gingival fibroblasts as well as a macrophage/epithelial cell co-culture model 32,43 and are significantly increased in patients with periodontitis. 44 Interleukin-6 was found to be induced by T. forsythia in the same co-culture model 43 as well as in human peripheral blood monocytes, the latter using heat-inactivated instead of viable bacteria.…”

“…Structure of the Tannerella forsythia O ‐glycan adapted from Friedrich et al . Red lines indicate the deletion of the terminal nonulosonic acid in the mutants T. forsythia ATCC 43037 ∆pseC and T. forsythia UB 4 ∆legC (1) and the three‐sugar truncation of the O ‐glycan in T. forsythia ATCC 43037 ∆wecC (2), respectively…”

“…In the T. forsythia ATCC 43037 type strain, it is present as a modified pseudaminic acid (Pse, 5,7-dia cetamido-3,5,7,9-tetradeoxyl-glycerol-manno-non-2-ulosonic acid), whereas isolate T. forsythia UB4 was found to carry a modified legionaminic acid (Leg, 5,7-diacetamido-3,5,7,9-tetradeoxyd-glycerodgalacto-non-2-ulosonic acid) instead. 29 These sugars belong to the structural class of nonulosonic acids, acidic nine-carbon (C9) α-keto sugars, which are best represented by the sialic acid family. 30,31 The two T. forsythia strains ATCC 43037 and UB4 vary not only in the terminal nonulosonic acid of the otherwise identical O-glycan, 29 but also in displaying different growth characteristics, monospecies biofilm formation and behavior in a multispecies biofilm consortium.…”

“…25 Both the T. forsythia S-layer and the attached glycan, especially the nonulosonic acids, were found to facilitate monospecies biofilm formation and the localization of T. forsythia within a multispecies biofilm. 25,29 The glycosylated S-layer of T. forsythia ATCC 43037 was shown to suppress the production of proinflammatory cytokines in U937 macrophages and human gingival fibroblasts, 32 to modulate the immune response through recognition by the macrophage inducible C-type lectin (Mincle) 33 and facilitate adhesion to and invasion of gingival epithelial cells. 22 Outer membrane vesicles of T. forsythia ATCC 43037, naturally carrying the intact glycosylated S-layer, were found to induce expression of TNFα and IL-8 in U937 macrophages, and IL-6, IL-8 and MCP-1 in primary human periodontal ligament fibroblasts.…”