Data_Sheet_1_A General Protein O-Glycosylation Gene Cluster Encodes the Species-Specific Glycan of the Oral Pathogen Tannerella forsythia: O-Glycan Biosynthesis and Immunological Implications.pdf

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.</p

B. longum 35624 EPS composition and structure.

<p>The structure is annotated as the chemical formula and in condensed form. Capital letters denote the residues as in Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162983#pone.0162983.g006" target="_blank">6</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162983#pone.0162983.g007" target="_blank">7</a>.</p

¹H and ¹³C NMR chemical shifts (δ, ppm) of the exopolysaccharide (recorded at 338 K) and the tetrasaccharide os211 (recorded at 300 K) from <i>B</i>. <i>longum</i> 35624.

<p>¹H and ¹³C NMR chemical shifts (δ, ppm) of the exopolysaccharide (recorded at 338 K) and the tetrasaccharide os211 (recorded at 300 K) from <i>B</i>. <i>longum</i> 35624.</p

<i>Bifidobacterium longum</i> general genome features.

<p><i>Bifidobacterium longum</i> general genome features.</p

EPS gene cluster.

<p>Illustration of the EPS cluster located in the <i>B</i>. <i>longum</i> <b>35624</b> genome and comparison to similar clusters located in <i>B</i>. <i>longum</i> 105-A, <i>B</i>. <i>longum</i> subsp. <i>longum</i> JCM1217 and <i>B</i>. <i>longum</i> subsp. <i>longum</i> NCC2705. Each gene is colour-coded according to function which is indicated in the legend located at the end of the page. Percentages represent the percent of sequence similarity at the protein level with corresponding genes in the <i>B</i>. <i>longum</i> <b>35624</b> genome. The locus tags of the first and last genes located in the EPS clusters of <i>B</i>. <i>longum</i> 105-A, <i>B</i>. <i>longum</i> subsp. <i>longum</i> JCM1217 and <i>B</i>. <i>longum</i> subsp. <i>longum</i> NCC2705 are also indicated in the illustration.</p

B. longum 35624 EPS proton and carbon signals.

<p>(<b>A</b>) A selected region of the multiplicity-edited, gradient enhanced ¹H, ¹³C-HSQC NMR spectrum of the exopolysaccharide. Letters denote the residues as given in the structural formula and arabic numerals denote the respective pyranose position. Resonances from anomeric carbons/protons, glycosylation sites and resolved signals are annotated. (<b>B</b>) Selected region of the ¹H, ¹³C-HSQC-TOCSY NMR spectrum (600 MHz) of the acid-treated <b>35624</b> EPS. Arabic numerals before and after oblique stroke denote carbons and protons, respectively.</p

B. longum 35624 EPS characterization.

<p>(A) The 600 MHz ¹H NMR proton spectrum of the acid-treated <b>35624</b> EPS (D₂O, 338 K) is illustrated. A part of the high-field region is displayed in the insert. <b>(B)</b> Expansion plot of the 150 MHz 13C NMR spectrum of the acid-treated <b>35624</b> exopolysaccharide. The anomeric signals on the left confirmed the presence of a hexasaccharide repeat unit.</p

Mild acid hydrolysis of EPS.

<p>(A) Separation of EPS fragments by PGC HPLC with MS/MS detection. The extracted ion chromatogram for mass 1008.39 Da shows four peaks. Their reducing end sugar was clearly revealed by ESI-MS/MS. Their assignment as either Gal or Glc and the interpretation in terms of fragment structures was done <i>a posteriori</i> based on MALDI-TOF data and on knowledge of the EPS structure. (B) Example of a MALDI-TOF/TOF fragment spectrum showing b-ions from the non-reducing and y- and y´ (= ^1,5x) -ions from the reducing end.</p

Phylogenetic tree based on the B. longum core-genome.

<p>(A) The <i>B</i>. <i>longum</i> subsp. <i>longum</i> phylogenetic group. (B) The <i>B</i>. <i>longum</i> subsp. <i>infantis</i> phylogenetic group. <i>B</i>. <i>longum</i> subsp. <i>longum</i> and <i>B</i>. <i>longum</i> subsp. <i>infantis</i> type strains are indicated in blue text. <i>Lactobacillus salivarius</i> was included as an outlier.</p

B. longum 35624 electron microscopy.

<p>(A) A layer of extracellular polysaccharide is clearly visible by electronic microscopy of the <b>35624</b> strain. (B) The isolated and purified EPS is illustrated.</p