Cell Wall and Amino Sugar Metabolism of Tannerella forsythia

1) Project title. Uncovering the cell wall and amino sugar metabolism of Tannerella forsythia Impact on the adaptation to the oral habitat 2) Content of research project. T. forsythia (Tf) is a pathogenic bacterium that causes periodontitis and inhabits oral biofilms. Its cell wall contains a typical peptidoglycan (PGN) layer a net-shaped polymer whose backbone is comprised of the amino sugars N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc) as well as unique glycoproteins displaying amino sugars and a sugar acid (nonulosonic acid, NulO). The apparent lack of the general bacterial synthesis pathways yielding the biosynthetic precursors of GlcNAc and MurNAc suggests that proliferation of Tf relies on their scavenging or retrieval form cohabiting bacteria or host tissues. The project intends to unravel the intriguing MurNAc-requirement of Tf, investigate a possible additional requirement for GlcNAc, elucidate the PGN synthesis pathway and analyse a cross-feeding with the synthesis of amino sugars and NulO of Tf glycoproteins. This knowledge is pivotal to understanding the bacteriums strategies to thrive in oral biofilms. 3) Hypotheses. (a) We have found recent evidence that a new MurNAc recycling route leading to metabolically active MurNAc (UDP-MurNAc) is elaborated in Tf. We will characterize key enzymes of this pathway, elucidate the retrieval of external MurNAc and MurNAc-containing PGN turn-over products, and unravel a link of PGN recycling and synthesis. (b) Based on our hypothesis of an unexplored UDP-GlcNAc synthesis route in Tf, we will investigate candidate enzymes and determine the role of external GlcNAc. (c) To obtain detailed insight into the cell wall metabolism, we will explore, if Tf can synthesize amino sugars de novo or if it relies on external sources - such as the biofilm promoting sugar acids or amino sugars of host cells - for cell wall precursor synthesis. Given the structural similarity of MurNAc and sugar acids, we aim at clarifying a possible connection between PGN synthesis and sugar acid metabolism. Eventually, we will explore in mixed biofilms how Tf PGN synthesis can benefit from cohabiting bacteria. 4) Methods. The proposed work is conceptuated as a joint project between C. Mayer (Universität Tübingen, DEU) and C. Schäffer (Universität für Bodenkultur Wien, AUT) and will exploit a complementary set of equipment and expertise in the fields of carbohydrate biochemistry, cell wall metabolism, radioisotope- labelling, and mass spectrometry (C. Mayer), glycobiology, glycan analytics, Tf genetics, cultivation of anaerobes, biofilm, and electron microscopy (C. Schäffer). X-ray crystallography structures of selected enzymes will be elucidated by a co-worker. 5) Explanation indicating what is new/special about the project. The results of this study will unravel new routes of bacterial cell wall and amino sugar metabolism which are valid beyond Tf and simultaneously pave new avenues for antibacterial treatment of periodontal diseases.

Periodontitis is a globally occurring, bacterial inflammatory disease of the teeth-supporting tissues. It is the major cause of tooth-loss and affects systemic diseases such as diabetes and Alzheimers disease. Periodontitis is linked to a multi-bacterial biofilm that develops on the surface of the teeth in anaerobic regions of dental pockets. A detailed understanding of the biofilm bacteria is important for the development of targeted strategies for the control and treatment of the infection. In this context, the cell wall metabolism of the oral pathogen Tannerella forsythia as a major driver of periodontitis was investigated. When grown in laboratory culture, the bacterium requires the cell wall sugar N-acetylmuramic acid as a growth factor; an intact cell wall is decisive for bacterial viability and pathogenicity. We found that T. forsythia is deficient in otherwise essential cell wall biosynthesis genes, which also translates into resistance to the antibiotic fosfomycin. We hypothesized that the bacterium has elaborated novel concepts for its cell wall metabolism in which cell wall components scavenged form cohabiting biofilm bacteria in the oral habitat might play a role. Using a combination of microbiological, genetic, biochemical, synthetic, analytic, and micros-copic approaches in a collaborative effort between research groups at Universität für Boden-kultur Wien and the Eberhard-Karls University, Tübingen, Germany, first, the composition of T. forsythias cell wall material (peptidoglycan) was elucidated and found to be of a wide-spread type, confirming the presence of N-acetylmuramic acid. Growth experiments upon provision of defined concentrations of N-acetylmuramic acid demonstrated the presence of a novel import protein for the sugar followed by conversion into a precursor for its incorporation into the cell wall, involving the subsequent activities of two novel enzymes. A distinct proportion of N- acetylmuramic acid is phosphorylated by another novel enzyme (MurK kinase) and channeled into the energy metabolism. In parallel, T. forsythia catabolizes larger cell wall fragments occurring during natural cell wall turn-over and emerging from decayed biofilm bacteria. The oral habitat was mimicked by supplementing T. forsythia cultures with defined cell wall fragments obtained after controlled enzymatic digestion and with intact peptidoglycan form various bacteria. We could identify strategies of T. forsythia for degradation, uptake and processing of external peptidoglycan to benefit its cell wall synthesis. For this, the membrane transporter AmpG was demonstrated to play an important role; for the transporter, a broader substrate range than that known for the E. coli counterpart was determined. This project delivered mechanistic insight into a novel concept of bacterial cell wall metabolism. Specifically, it unraveled in T. forsythia the presence of essential novel enzymes and transporters of unexpected specificity. These might constitute novel targets for urgently needed innovative treatment concepts of periodontitis. Thus, the outcome of this project has applicability beyond basic science.