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

Markus B Tomek,Irene Nimeth,Christina Schäffer,Laura Neumann,Arun Everest-Dass,Heinz Himmelbauer,Bettina Janesch,Markus Windwarder,Valentin Friedrich,Nikolaus F Zwickl,Kristina Fuchs,Daniel Maresch,Juliane C Dohm,Daniel Kolarich,Friedrich Altmann

doi:10.3389/fmicb.2018.02008

Markus B Tomek, Irene Nimeth + Show 13 more

Open Access

https://doi.org/10.3389/fmicb.2018.02008

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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.

Highlights

Protein glycosylation in bacteria is a frequent modification of secreted and cell-surface proteins, such as flagella, pili, autotransporters, and surface (S-) layer proteins (Upreti et al, 2003; Schäffer and Messner, 2017)
An alignment of protein O-glycosylation gene clusters from different T. forsythia strains for which genome sequences were publically available was generated using the software MultiGeneBlast 1.1.13 (Camacho et al, 2009; Medema et al, 2013) in “homology search” mode
A partial protein O-glycosylation gene locus (Posch et al, 2011; Coyne et al, 2013) as well as genes for the biosynthesis of cytidine-5 -monophosphate (CMP)-pseudaminic acid (Pse), which is the activated form of this sugar acid required for its incorporation into the S-layer glycan, (Friedrich et al, 2017), were identified in the genome of T. forsythia ATCC 43037

Summary

Introduction

Protein glycosylation in bacteria is a frequent modification of secreted and cell-surface proteins, such as flagella, pili, autotransporters, and surface (S-) layer proteins (Upreti et al, 2003; Schäffer and Messner, 2017). General protein glycosylation systems are employed, yielding a suite of proteins with diverse locations and functionalities that carry one or more copies of an identical glycan (Schäffer and Messner, 2017). The genetic information governing protein glycosylation is frequently organized in protein glycosylation gene clusters (Nothaft and Szymanski, 2010), which encode nucleotide sugar pathways genes, genes for Gtfs, glycan processing and modifying enzymes, ligases, and transporters. Most protein O-glycosylation systems investigated so far secrete virulence factors or translocate glycoproteins to the bacterial cell surface, exemplified with Campylobacter spp. Most protein O-glycosylation systems investigated so far secrete virulence factors or translocate glycoproteins to the bacterial cell surface, exemplified with Campylobacter spp. (Szymanski et al, 1999, 2003), Neisseria spp. (Ku et al, 2009; Vik et al, 2009; Hartley et al, 2011), Bacteroides spp. (Fletcher et al, 2009), Actinomycetes (Espitia et al, 2010), Francisella tularensis (Egge-Jacobsen et al, 2011), Acinetobacter spp. (Iwashkiw et al, 2012; Lees-Miller et al, 2013; Harding et al, 2015), Burkholderia

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