Tannerella S-layer nanoglycobiology for drug development

Tannerella forsythia (Tf) is a Gram-negative, filamentous oral anaerobe that has been identified as a crucial periodontal pathogen. Periodontitis is one of the most common inflammatory diseases amongst the adult population, and, in its chronic form, it is the principal cause of tooth loss. Tf infection may also exert a long-term impact on systemic health, such as cardiovascular disease or arteriosclerosis. Consequently, there is a great biomedical interest in understanding the bacteria-host cross-talk that forms the basis of health, disease, and healing. As a basis for determining microbial pathogenesis of Tf infection, the virulence factors involved in the steps of pathogenesis will have to be identified and characterized. Preliminary studies indicate that the outermost crystalline surface layer (S layer) of Tf is a potential virulence factor, because functional tests revealed that it is involved in inducing hemagglutination and adherence/invasion of epithelial cells. We want to investigate this aspect in the light of the recent finding that the S-layer of Tf is glycosylated, because accumulated knowledge on bacterial disease pathogenesis revealed that glycosylated surface molecules may play pivotal roles in bacterial adherence to specific tissues or in triggering of a host immune response. Biomedical research over the past years has shown that especially carbohydrates possess an enormous potential as lead structures for drug discovery. As a first step in understanding the importance of the S layer glycoproteins to the Tf-host crosstalk, we propose to investigate the glycobiology of the Tf S layer. This is especially interesting, because Tf is the only Gram-negative bacterium that is currently known to possess a glycosylated S-layer, which, in addition, seems to be structurally unique, because it appears to consist of two different glycoproteins. We hypothesize that the glycan(s) attached to the Tf S-layer proteins, representing the first cell surface structures that are encountered by the host, are involved in mediation of virulence. As a way to characterize the Tf S-layer glycobiology and to test our hypothesis, our research goals include: A) determination of the fine structure and composition of the Tf S layer glycoprotein lattice, B) identification of the anchoring mechanism of the Tf S layer to the cell wall, C) in-depth analysis of the two S layer glycoproteins with regard to glycan structure(s), glycosylation site(s), glycan chain length, and mass as well as of the biosynthetic machinery underlying S-layer glycosylation, and D) performance of first cell culture studies on the contribution of S layer glycosylation to the Tf virulence potential. Detailed studies on the glycobiology of the Tf S layer may reveal novel pathogenic strategies in Gram-negative organisms, which, in the future, may constitute new targets for interfering with the pathogen`s ability to establish infection in periodontal disease. While unraveling the S layer glycobiology will appear mainly to benefit basic science, translational researchers and clinicians will subsequently be able to incorporate those findings to improve care and disease prevention.

Periodontitis is a bacterially-induced, multifactorial, inflammatory disease of the periodontium affecting millions of people worldwide. In its chronic form, it is the major cause of tooth loss and it can also impact systemic health. Despite distinct bacteria have been identified as key triggers of periodontitis, the etiology of the disease is still poorly understood. Based on the recognition that cell surface-displayed carbohydrates are important elements in defining the life of pathogenic bacteria, and, thus, lead structures for drug development, this project aimed at unravelling the cell surface glycobiology of the periodontal pathogen Tannerella forsythia. The basis for the design of this project were reports on the presence of a 2D crystalline cell surface layer (S-layer) covering T. forsythia cells and indications for its modification with carbohydrates. We hypothesized that the glycan(s) attached to the S-layer, representing the outer-most cell surface structures facing the environment, are involved in mediation of virulence by participating in the bacterial life-style, such as colonization of the oral habitat and biofilm formation, and in host-pathogen interactions. The major achievements of this project were: (i) Demonstration that the T. forsythia S-layer is an important virulence factor that attenuates the host immune response to this pathogen by evading the bacteriums recognition by the innate immune system. (ii) Microscopic elucidation of the S-layer ultrastructure at the nanometer scale revealing the so far unique situation of two intercalating S-layer glycoproteins co-assembling into a single, ~22-nm thick, S-layer. (iii) In-depth analysis of S-layer glycosylation in T. forsythia, revealing a general protein O-glycosylation system that is essential for creating a rich glycoproteome pinpointing a possible relevance for the virulence of this bacterium. (iv) Proposal of a phylum-wide protein O-glycosylation system based on experimental evidence, allowing exchange of glycosylation modules between bacteria for the future determination of glycan structure- function relationships. (v) Analysis of the T. forsythia lipopolysaccharide, indicating that this bacterium possesses a novel, rough-type lipopolysaccharide which stimulates human immune cells to produce proinflammatory mediators in a dose-dependent manner; the requirement of serum cofactors makes it different from the lipopolysaccharides of other periodontal bacteria. Periodontal disease remains a critical issue in public health and presents a rich field for future research. Fuelled by this project it is evident that knowledge of oral glycobiology contributes to a better understanding of the pathogenesis implicated by periodontal infection as well as to the identification of possible molecular targets for novel diagnostic tools and therapeutic interventions. The outcome of this project has substantial potential in applications beyond basic science, as needed in pharmaceutical and biotech industry to improve health care and disease prevention.