S-layer (glycosylation) impact on Tannerella biofilm

The investigation of the pathogenic mechanisms employed by key periodontal pathogens is of biomedical relevance. Progression of oral disease is often dependent upon accumulation of oral plaque, a prime example of a bacterial biofilm. Establishing the mechanisms involved in formation and progression of both mono- and multispecies oral biofilms may pinpoint new targets for interfering with the pathogens` ability to establish biofilm formation and, eventually, infection in periodontal disease. T. forsythia is an anaerobic, oral pathogen classified as a late colonizer of multispecies-gingival plaque biofilms that constitutes the focus of this research project. It was shown at the host research institution that T. forsythia possesses a so far unique cell envelope that is represented by a 2D crystalline S-layer formed by co-assembly of two S layer glycoproteins (TfsA, TfsB) which are modified with distinct oligosaccharides. This is of special interest considering the importance of bacterial cell surface structures for the development and/or architecture of biofilms. In fact, the S-layer glycoproteins and a predicted export system are up-regulated under biofilm conditions. Our preliminary data indicate that TfsB reduces intraspecies cell aggregation while enhancing surface attachment; a defined truncation of the S-layer glycan resulting in loss of net negative charge promotes biofilm formation. This indicates a distinct regulation between S-layer biosynthesis, glycosylation and biofilm formation. Investigating the influence of the cell surface structures of T. forsythia on biofilm formation is an ideal starting point to gain a more detailed insight into the complex scenario of oral biofilm communities. We hypothesize that the T. forsythia S layer and its glycosylation status, representing the immediate contact zone of the bacterium with its environment, affect the bacterial intra- and inter-species communication in the oral cavity as a prerequisite for the biofilm lifestyle, and, consequently, the ability to establish periodontal infection. Investigating the biochemical and molecular basis for this phenomenon, will increase our understanding of the role of T. forsythia in periodontal disease. As a way to test our hypothesis, the research goals of this proposal are 1) determination of optimal biofilm conditions for T. forsythia wild-type cells, testing different substrates and environmental factors; 2) investigation of the effect of the T. forsythia S layer, its glycosylation and specific components thereof on biofilm formation with a focus on the initial attachment step; 3) analysis of the impact of the T. forsythia S-layer (glycosylation) on the development of multispecies biofilms. Respective T. forsythia S-layer and glycosylation mutants (focusing on the modified pseudaminic acid a frequent adhesin of pathogenic bacteria - and the N acetylmannosaminuronic acid residues of the S-layer glycan, both contributing to a net charge of the bacterial cell surface) as well as recombinant S layer proteins and purified S layer glycan are available.

Tannerella forsythia (Tf) is an oral pathogen that along with Porphyromonas gingivalis and Treponema denticola forms the so-called red complex, a group of species involved in periodontitis. It is assumed that bacteria within the oral cavity exist predominantly in surface- associated communities called biofilms, to which in the oral cavity it is referred as plaque. Living within a biofilm provides a survival advantage for microorganisms, especially for pathogens. Biofilms are usually composed of several species that interact with each other (synergy or antibiosis). The cells become embedded in a matrix, a self-produced glue-like substance, which serves as protection against environmental influences. In addition, the matrix of biofilms from pathogens delays the immune response of the host and limits the access of antibiotics. Understanding the mechanism of microbial biofilm formation in the oral cavity is therefore important for developing agents that target the oral biofilm to contribute to the treatment of periodontitis.For the formation of mono- and multispecies biofilms, the bacterial cell envelope plays an important role, since it is the immediate contact zone of the bacteria with the environment. Tf is an interesting model organism because of its unique cell envelope. Tf is a Gram-negative bacterium covered with a surface (S)-layer. The S-layer is composed of two types of O- glycosylated proteins, TfsA and TfsB. In our laboratory, single and double S-layer mutants are available. Additionally, we have in hands a ?wecC mutant, which lacks three charged sugar residues at the end of the S-layer O-glycan. This allows us to perform comparative analyses and to assess the influence of the S-layer and its glycosylation status on biofilm formation. Other studies demonstrated that Tf wild-type is a rather weak biofilm former, whereas the ?wecC mutant was found to readily form biofilms. Interestingly, it was reported assert the ?wecC mutant is hydrophobic, which could not be confirmed in our experiments. After optimizing the conditions, Tf wild-type and the ?wecC mutant showed increased biofilm formation. This was of particular relevance for our research, since for a precise analysis a certain degree of efficiency is required, especially for the detection of weaker biofilm. The detailed analysis of the ?wecC mutant revealed hydrophilic cells with a higher wettability. They auto-aggregate at a higher rate and consequently show higher biofilm formation. The degree of aggregation is low for the S-layer mutants, but the affinity to substrates/surfaces are very high. This leads to a carpet-like biofilm with two or three cell layers without 3- dimensional structures or cell-clusters.