Tannerella general protein O-glycosylation and pathogenicity

The anaerobic oral biofilm-forming pathogen Tannerella forsythia (Tf) is one of the main etiological agents of periodontitis. Because of the increasing frequencies with which bacterial glycosylation systems are seen in pathogenic species, they have come under enhanced scrutiny; especially the "sweet" cell envelope of pathogens contributes to their cross-talk with the hosts. Consequently, for the study of virulence mechanisms and the associated development of therapeutic strategies, a molecular understanding of the glycans displayed in the cell envelope of pathogens is necessary. Although a complete genome sequence is available for Tf, relatively little is known about the cell biology and glycobiology of this pathogen. Tf is the only Gram-negative bacterium known to possess a 2D crystalline glycoprotein layer (S-layer) as outermost cell envelope layer. The Tf S-layer is a virulence factor and contributes to delaying the host immune defense. It is evident that the uniqueness of the Tf cell envelope is defined by its glyco-biology (ongoing FWF- project). The S-layer is unique in being formed by co-assembly of two different proteins, which are modified with a specific type of complex oligosaccharides a multiple sites. A novel type of rough lipopolysaccharide is predicted to anchor the S-layer glycoprotein assembly to the outer membrane, and the Tf outer membrane is dominated by additional antigenic, high molecular-mass glycoproteins. Some of these as well as the S-layer glycoproteins are up- regulated in the biofilm life-style of Tf. The present research proposal is designed to get insight into the Tf S-layer O-glycosylation system and its scope at the molecular level. We have evidence that several Tf proteins are modified with this specific S-layer O-glycan, pinpointing to a glycosylation scenario of general relevance. We hypothesize that the S-layer glycosylation system of Tf affects a wide number of different target proteins and, thus, is of high relevance for the bacterium. Considering the presence of a modified pseudaminic acid residue in the Tf S-layer glycan, it is conceivable that this glycan does not only have a demonstrated impact on the bacterium`s life style and, hence, development of periodontal disease, but also is a mediator of pathogenicity. As a way to profile this O-glycosylation system to assess its contribution to bacterial pathogenicity, our research goals are: A) molecular characterization of key modules of the Tf S-layer protein glycosylation; B) assessment of the influence of cell envelope glycosylation on the immune response elicited against Tf; and C) investigation of the "glyco-impact" on the life style of Tf by analyzing biofilm formation of Tf mutants with defined defects in selected genes of the glycosylation system. Profiling of this general O-glycosylation system of Tf at the molecular level and investigating how it affects the physiology of Tf may reveal novel pathogenic strategies in Gram-negative organisms. This, in the future, may constitute new targets for interfering with the pathogen`s ability to establish infection in periodontal disease and for developing of novel diagnostic tools.

The study how bacterial pathogens cause disease in man is a timely research subject of medical importance. By understanding the molecular basis of the processes governing pathogenicity, it will be possible to design rational approaches for controlling or even preventing diseases. Periodontitis continues to be the most frequent inflammatory disease world-wide. In its chronic form, it is the major cause of tooth loss and has a possible impact on systemic health. While distinct bacteria have been identified as key triggers of periodontitis, the pathogenesis of the disease is still poorly understood. Tannerella forsythia, thriving in an oral biofilm known as dental plaque, is recognized as a periodontal pathogen. We found that this bacterium displays at its surface thousands of copies of a unique, complex dekasaccharide bound to specific surface proteins, which are known virulence factors. Intriguingly, the dekasaccharide is modified with a terminal nonulosonic acid; this is a mimic of sialic acid for which a variety of biological functions are known. This let us hypothesize that the protein-linked dekasaccharide underpins T. forsythia's pathogenicity. Considering that cell surface glycans possess an enormous potential as lead structure for drug discovery, this project aimed at providing insight into how conserved the dekasaccharide is among clinical T. forsythia isolates, how it is synthesized by the bacterium, how it impacts biofilm formation, and how it is active at the immune interface.Achievements of this project were: (i) Discovery of a mismatch between available T. forsythia ATCC type strain and deposited genome sequence, followed by sequencing of the type strain. (ii) Demonstration that the genetic information for dekasaccharide synthesis is encoded by a polycistronic 27-kb glycosylation gene cluster. (iii) Bioinformatic identification of that gene cluster in all publically available T. forsythia genomes, but not in a health-associated isolate. (iv) Identification and characterization of key enzymes for the step-wise assembly of the T. forsythia-specific part of the dekasaccharide, with a focus on nonulosonic acid. (v) Discovery of a new secretion system in T. forsythia employed for the export of cell surface glycoproteins. (vi) Discovery of T. forsythia outer membrane vesicles containing proinflammatory cargo. (vii) Identification of a novel T. forsythia fucosidase effective in procuring substrates from host glycoproteins. (viii) Extension of the glycobiology perspective of the T. forsythia cell wall by the identification of novel glycolipids. (iix) Demonstration that the dekasaccharide modulates immune cell effector functions and thereby T. forsythia's persistence in the host, as well as (ix) T. forsythia's biofilm formation capability.This project demonstrated that the cell surface dekasaccharide underpins the pathogenicity of T. forsythia and serves as a signature of disease-associated T. forsythia strains. Thus, the project provided possible molecular targets for novel diagnostic tools and therapeutic interventions. This has substantial potential in applications beyond basic science, as needed in pharmaceutical and biotech industry to improve health care and disease prevention.