Glycobiology of the oral bacterium Selenomonas sputigena

Periodontitis continues to afflict the majority of the worlds population as the major cause of tooth loss. It is an inflammatory disease of the periodontium triggered by a polymicrobial oral biofilm. Current efforts to unravel the still not fully understood etiology of periodontitis focus on the interplay that occurs among the numerous oral bacteria in the biofilm community. The structural, functional, and molecular properties of cell surface glycoconjugates support a key role of these molecules in inter-bacterial communication and recognition. Supported by own data on the glycobiology of the periodontopathogen Tannerella forsythia (projects P20605 and P24317), and immunological research by others, we hypothesize that distinct oral bacteria possess a diverse, yet to be discovered, glycome and that their cell surface glycobiology is a hallmark of polymicrobial community inter-actions and relevant to the development and the properties of dental plaque. Despite its possible association with periodontitis, Selenomonas sputigena (Selspu), a Gram-negative, multi- flagellated, motile, anaerobic rod, is almost uninvestigated. Based on recent findings from our laboratory we hypothesize that Selspu employs various glycobiology-based strategies for oral colonization and survival, because: (i) Selspu possesses a diverse glycoproteome as inferred from positive carbohydrate staining and lectin reactivity of whole cell extracts after separation by SDS-PAGE; (ii) There are indications that Selspu possesses glycosylated flagella; these might be important as both colonization and pathogenicity factor; (iii) Selspu is capable of co-aggregating with T. forsythia, pinpointing a possible association with the effects of the red-complex bacteria, and (iv) The Selspu genome provides rich glycobiology information. Especially the prediction of uncommon sugar residues such as L-fucose (an immunogenic sugar), pseudaminic acid (occurring on the flagella of pathogens and on T. forsythia proteins), -O-acetylglucosamine (rarely found on bacterial proteins), and 4-amino-arabinose (known as lipid A modification required for resistance to antimicrobial peptides) makes Selspu a prime glycobiology candidate. To get insight into the glycobiology of Selspu as a basis for assessing its role in the development of dental plaque, our research goals are: A) Identification of prominent Selspu glycoproteins and determination of their glycan composition and structure; B) Analysis of flagella glycosylation; C) Structural investigation of the Selspu lipopolysaccharide; and D) Analysis of the Selspu biofilm matrix focusing on a putative exo-poly- saccharide. These work packages will be accompanied by transcription analysis of predicted genomic Selspu glycosylation loci and analysis of selected Selspu carbohydrate-active enzymes. This project contributes to deciphering glycobiology aspects of an oral bacterium associated with dental plaque and can inform about bacterial strategies relevant to the pathogenesis of periodontitis. In the future, these may constitute new targets for interfering with the oral microbial communitys ability to establish infection in periodontal disease.

Because of the worldwide spread of antimicrobial resistances, there is an urgent need of novel drugs to be fueled into the development pipeline. Sugar chains linked to the cell surface of pathogenic bacteria have an enormous potential as lead structures for the design of such novel drugs. The focus of our research is on deciphering the cell surface glycobiology of oral pathogens in the context of periodontitis. Periodontitis is a globally important inflammatory disease of polymicrobial etiology, which is still not fully understood. Periodontitis is, in its chronic form, the major cause of tooth loss, and, according to recent data, also impacts systemic health. This project deals with the little investigated bacterium Selenomonas sputigena, whose pathogenicity status is unknown, but which we found to coaggregate with the demonstrated oral pathogen Tannerella forsythia. Overall, this project aimed at providing new insights into the sugar-containing compounds that are displayed at the surface of these two oral bacteria and how these compounds might impact the bacterias` pathogenicity. For this purpose, a portfolio of microbiological, biochemical and immunological techniques, accompanied by the development of tailored methods was applied. Achievements of this project were: (i) Discovery of a flagellin protein modified with sugar chains (glycoprotein) as the most abundant glycoprotein of S. sputigena and demonstration that this glycoprotein contains many copies of hitherto unknown sugar chains. The presence of a sugar component that is not found in humans makes this glycoprotein a candidate for the design of an anti-infective strategy. (ii) S. sputigena was found to possess a sugar-lipid composite (lipopolysaccharide) of novel composition in its outer cell membrane; this showed a high immunestimulatory activity and, thus, might justify the classification of S. sputigena as an oral pathogen. (iii) A comparative analysis of the genomes of cohabiting T. forsythia stains revealed that a glycosylation gene cluster encoding, among others, genes for the biosynthesis of a class of sugar acids that is typical of many pathogens (nonulosonic acids), is a hallmark of periodontitis-associated T. forsythia strains. (iv) These sugar acids serve as decoration of cell surface glycoproteins and impact the establishment of T. forsythia in a multispecies oral biofilm model mimicking the native situation in the oral habitat. (v) The nonulosonic acids further contribute to dampening the response of host cells and, hence, play a pivotal role in orchestrating the bacteriums virulence by ensuring its persistence in the host. This project contributes to increasing our understanding of the under-investigated surface properties of oral pathogens. Our findings translate into a potential role of the cell surface sugar structures of both S. sputigena and T. forsythia in the virulence of these species when interacting with host tissues and the immune system, from within or beyond the biofilm. Thus, we 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.