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 <i>Tannerella forsythia</i> is heavily glycosylated with a unique, complex decasaccharide that is <i>O</i>-glycosidically linked to the bacterium's abundant surface (S-) layer, as well as other proteins. The S-layer glycoproteins are virulence factors of <i>T. forsythia</i> and there is evidence that protein <i>O-</i>glycosylation underpins the bacterium's pathogenicity. To elucidate the protein <i>O</i>-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 <i>O-</i>glycans, we show that the gene cluster encodes the species-specific part of the <i>T. forsythia</i> ATCC 43037 decasaccharide and that this is assembled step-wise on a pentasaccharide core. The core was previously proposed to be conserved within the <i>Bacteroidetes</i> phylum, to which <i>T. forsythia</i> is affiliated, and its biosynthesis is encoded elsewhere on the bacterial genome. Next, to assess the prevalence of protein <i>O-</i>glycosylation among <i>Tannerella</i> sp., the publicly available genome sequences of six <i>T. forsythia</i> 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 <i>Tannerella</i> 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 <i>T. forsythia</i>'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 <i>O-</i>glycan is pivotal to modulating DC effector functions, with the <i>T. forsythia</i>-specific glycan portion suppressing and the pentasaccharide core activating a Th17 response. We conclude that complex protein <i>O-</i>glycosylation is a hallmark of pathogenic <i>T. forsythia</i> strains and propose it as a valuable target for the design of novel antimicrobials against periodontitis.