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

Abstract

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.

Keywords: S-layer; carbohydrate-active enzymes; glycosyltransferase; immunogenicity; locus for glycosylation; methyltransferase; periodontitis.

FIGURE 1

(A) Scheme of the T. forsythia ATCC 43037 S-layer O-glycan structure. Monosaccharide symbols are shown according to the Symbol Nomenclature for Glycans (SNFG) (Varki et al., 2015). Please note that the position of the branching Fuc remained unclear (Posch et al., 2011) until it was determined in the course of this study to be on the reducing-end Gal. (B) Scheme of the 27-kb protein O-glycosylation gene cluster of T. forsythia ATCC 43037. Wzx (black), flippase; pseBCFHGI (green), CMP-Pse biosynthesis genes; gtfSMILE (blue), Gtf genes; mtfJOY (yellow), Mtf genes; asnB (putative asparagine synthetase B), wecC (UDP-N-acetyl-D-mannosamine dehydrogenase) and wecB (UDP-N-acetylglucosamine 2-epimerase) (purple); hypothetical proteins, HP (gray). Genes are not drawn to scale.

FIGURE 2

Alignment of protein O-glycosylation gene clusters from different T. forsythia strains showing comparable sizes and gene organizations (drawn to scale). Genes showing sequence identity > 50% and sequence coverage > 50% between strains appear in the same color. The major difference in the analyzed strains are for genes synthesizing either CMP-Pse (pseBCFHGI; light green colors; strains ATCC 43037 and UB20) or CMP-Leg (legBCHIGF, ptmE; dark green colors; strains FDC 92A2, UB4, KS16, UB22). Genes encoding Gtfs (gtfSMILE; blue color), Mtfs (mtfJOY; yellow color) and carbohydrate modifying enzymes (asnB, wecC, wecB; gray color) show high sequence homology between analyzed strains. Genomes of all strains synthesizing CMP-Leg encode an additional putative Mtf gene (mtfX), which does not share sequence homology to other Mtfs located within the cluster. In strain UB22, mtfJ is not predicted and for strain 3313 only five out of seven genes needed for the synthesis of CMP-Leg are predicted confidently. Due to low homology, isolate Tannerella sp. HOT-286 (phylotype BU063) could not be aligned with the other T. forsythia strains; for that isolate, the genomic area between a wzx-like gene and the gtfE gene is shown for comparison. P, Pse transferase; L, Leg transferase; HP, hypothetical protein; the star symbol (^∗) indicates a transposable element; genes written in bold letters were investigated in detail in course of this study.

FIGURE 3

(A) Coomassie Brilliant Blue staining of crude cell extracts from T. forsythia ATCC 43037 wild-type and glycosyltransferase-deficient mutants after separation on a 7.5% SDS-PA gel. The S-layer glycoproteins (labeled TfsA and TfsB) are indicated and the downshifts resulting from glycan truncation can be seen in the mutants. S-layer glycoprotein bands were further processed for MS analyses. PageRuler Plus Prestained Protein Ladder (Thermo Fisher Scientific) was used as a protein molecular weight marker. (B) Western-blots probed with α-TfsA and α-TfsB antiserum for confirmation of the identity of S-layer glycoproteins. Glycoproteins from all glycosyltransferase-deficient mutants (ΔgtfSMILE) experienced a downshift resulting from glycan truncation, whereas the reconstituted strains (denoted with +) regained wild-type migration, indicating the presence of the complete mature glycan, proving successful recombination. (C, i–vi) ESI-MS sum spectra of β-eliminated TfsB O-glycans from T. forsythia wild-type and mutants. The glycan structures of the signals corresponding to the largest mass (bold m/z values) are shown in SNFG representations (Varki et al., 2015). O-Glycan signals detected for the respective mutants were assigned based on the m/z mass differences corresponding to the loss of individual sugar units and/or modifications.

FIGURE 4

Positive mode CID tandem spectra of reduced O-glycans from glycoproteins in T. forsythiaΔTanf_01300. (A) The m/z = 930.3¹⁺ precursor ion corresponds to the glycan composition Gal + GlcA + Xyl + Dig + Fuc₂ containing an ammonium adduct [M+NH₄]⁺. Its product ion spectra contained several typical Y/Z and a few B/C ion cleavages that were highly informative to sequence the proposed glycan structure. For example, the C_2βY_2β (m/z = 341) and B_2βY_2β (m/z = 322.9; B) ions clearly show that the GlcA is linked to a Fuc at the non-reducing end. Similarly, the reducing end Y_1βY_1γ double cleavage ion of m/z = 329 is indicative of a Fuc residue linked to the reducing-end Gal. Furthermore, this product ion spectra allows to clearly distinguish the presence of the additional fucose residue, which is lacking in the product ion spectra of m/z 784.37¹⁺ (B). Unique fragment ions (m/z = 329 and 651; bold) additionally supporting the location of the additional Fuc residue are only observed in the product ion spectra of m/z = 930.3¹⁺. (B) Product ion spectra from [M+NH₄]⁺ ions of m/z = 784.37 corresponding to the composition Gal + GlcA + Xyl + Dig + Fuc. Monosaccharide symbols are according to the Symbol Nomenclature for Glycans (SNFG) (Varki et al., 2015).

FIGURE 5

ESI-MS sum spectra of β-eliminated TfsB O-glycans from T. forsythia ATCC 43037 methyltransferase knock-out mutants. The glycan structures of the signals corresponding to the largest mass (bold m/z values) are shown in SNFG representation (Varki et al., 2015). Other O-glycan signals detected for the respective mutants were assigned based on the m/z mass differences corresponding to the loss of individual sugar units and/or modifications. The lack of methyl modifications is indicated by a red circle in the symbolic O-glycan structure representation.

FIGURE 6

Effects of protein O-glycosylation on T. forsythia immunogenicity. (A) Secretion of inflammatory cytokines by human DCs upon stimulation with T. forsythia wild-type (WT) and glycosyltransferase-deficient mutants (T. forsythia ΔgtfE, ΔgtfI, and ΔgtfS) as measured in culture supernatants by ProcartaPlex Multiplex Immunoassay. (B,C) T cell-priming upon APC stimulation with T. forsythia wild-type and glycosyltransferase-deficient mutants was assessed by culturing PBMCs. (B) T cell activation was measured via expression of CD25 by flow cytometry. Cells were pre-gated for live CD3⁺ cells, T cell proliferation was assessed after 8 days by CFSE dilution,. (C) CD4⁺ T cell differentiation was assessed by expression of signature transcription factors for Th17 (RORγT) and Treg (FoxP3) cells as measured by flow cytometry. All data are presented as mean ± SEM of triplicate determinations. One representative out of three (A) and two (B,C), respectively, independent experiments is shown. Statistically significant differences in (A) are indicated as ^∗p < 0.05 and ^∗∗∗p < 0.001 (Student’s t-test). LPS, E. coli O111:B4 LPS.

FIGURE 7

Model for the biosynthesis of the species-specific portion of the T. forsythia ATCC 43037 O-glycan. Upon synthesis of the pentasaccharide core on an undP lipid carrier, the first carbohydrate residue of the species-specific glycan is a Fuc residue conferred by GtfE. The glycan is elongated with a Gal residue which is transferred by GtfL and methylated by MtfY. The assembly of the three sugar branch, consisting of a ManNAcA residue (transferred by GtfI), a ManNAcCONH₂ residue (GtfM), which is methylated by either MtfJ or MtfO, and a Pse5Am7Gra residue (transferred via GtfS), completes the synthesis of the decasaccharide.