Carbohydrate-active enzymes in S-layer protein glycosylation

Background.

The past decade has been associated with remarkable discoveries revealing in vivo functions of glycans and the mechanistic paradigms by which glycans participate in physiology and contribute to disease pathogenesis. Recent findings indicate therapeutic benefits of specific glycan deficiency states in the pathogenesis of disease. Accumulated knowledge on bacterial disease pathogenesis revealed that different glycosylated surface molecules, such as lipopolysaccharides, capsules or glycoproteins may play roles in bacterial adherence to specific tissues or in triggering of a host immune response (O'Brien-Simpson et al., 2004). The finding that several human pathogens contain glycoproteins as surface decoration has opened up a rapidly developing area of biomedical research. Investigations over the past years have shown that carbohydrates especially possess an enormous potential as lead structures for drug discovery, aiming at antagonizing the interaction of the physiological carbohydrate ligands with their receptor proteins.

In the light of these findings, the presence of a glycosylated surface layer (S layer) protein on the oral pathogen Tannerella forsythia (Higuchi et al., 2000; Lee et al., 2006) is attracting attention. T. forsythia is a filamentous, Gram-negative, oral anaerobe, which has been implicated in the development of periodontitis, and whose genome sequence has been recently released. It is frequently found associated with other periodontopathogens in an oral biofilm, together constituting the “red complex” which is implicated in the severity of the disease. 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. The “red complex bacteria” are expressing numerous virulence factors that allow them to colonize the subgingival space, disturb the host’s defense system, invade and destroy the periodontal tissues, and, additionally, promote the immunodestructive response of the host (Holt & Ebersole, 2005). There is a great biomedical interest in understanding the host-bacteria cross-talk that forms the basis of health, disease, and healing.

There is clear evidence that the S layer of T. forsythia is a virulence factor. We hypothesize that the glycan(s) attached to the S-layer proteins, representing the outermost cell surface structures facing the environment, are involved in mediation of virulence. In an ongoing project, detailed studies on the S layer glycoprotein glycans of T. forsythia, focusing on the large-scale cultivation of the organism, on the establishment of isolation and purification protocols of the glycoproteins, and on the elucidation of the glycan structures are being performed.

Aims and methods.

This project is aiming at the functional characterization of the enzymes involved in the glycosylation process of the S layer proteins TfsA and TfsB of T. forsythia, leading eventually to the elucidation of a complete pathway for S layer protein glycosylation (compare with Steiner et al., 2008a). This information 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. In addition, it is important to characterize bacterial antigens of periodontopathic bacteria for further evaluation of their potential as vaccine candidates for a multispecies vaccine for periodontitis as an adjunct to current periodontal therapies.

The chosen approach will include: (i) based on the known biosynthetic pathways of nucleotide sugar precursors for the individual sugar residues present in the S layer glycan(s), degenerate primers will be designed to identify the corresponding sequences for the involved enzymes in the T. forsythia genome (this approach is necessary, because most of the ORFs of the genome are not annotated); (ii) chromosome walking starting from the identified site to identify a putative S layer glycosylation (slg) gene cluster (in all bacteria investigated so far, S layer glycosylation has been found to be encoded by an slg gene cluster (Messner et al., 2008)); (iii) in case no slg cluster is present, individual biosynthesis enzymes will be identified based on their known linkage-specificity by a bioinformatics approach, including CAZy database; (iv) cloning and overexpression of the glycosylation active enzymes; (v) set-up of functional enzyme assays starting from synthetic lipid-linked saccharides; (vi) characterization of the products of the enzymatic reactions by MS and NMR approaches; (vii) performance of kinetic studies of selected key modules of S layer protein glycosylation; (viii) structural analysis of those enzymes by CD and FT-IR spectroscopy, (ix) molecular modeling of key enzymes as well as simulation of enzyme-substrate recognition events, (x) crystallization of carbohydrate active enzymes and co-crystallization with substrates (Steiner et al., 2008b).

Higuchi, N., Murakami, Y., Moriguchi, K., Ohno, N., Nakamura, H., Yoshimura, F. (2000) Localization of major, high molecular weight proteins in Bacteroides forsythus. Microbiol. Immunol. 44, 777-780
Holt, S. C., Ebersole, J. L. (2005) Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia: the "red complex", a prototype polybacterial pathogenic consortium in periodontitis. Periodontology 2000 38, 72-122
Lee, S. W., Sabet, M., Um, H.S., Yang, J., Kim, H. C., Zhu, W. (2006) Identification and characteri¬zation of the genes encoding a unique surface (S-)layer of Tannerella forsythia. Gene 371, 102-111
Messner, P., Steiner, K., Zarschler, K., Schäffer, C. (2008) S-layer nanoglycobiology of bacteria. Carbohydr. Res. 343, 1934-1951
O‘Brien-Simpson, N. M., Veith, P. D., Dashper, S. G., Reynolds, E. C. (2004) Antigens of bacteria associated with periodontitis. Periodontology 2000 35, 101-134
Steiner, K., Novotny, R., Werz, D. B., Zarschler, K., Seeberger, P.H., Hofinger, A., Kosma, P., Schäffer, C., Messner, P. (2008a). Functional characterization of S layer glycoprotein glycan biosynthesis in Geobacillus stearothermophilus NRS 2004/3a. J. Biol. Chem. 283, 21120-21133
Steiner, K., Wojciechowska, A., Schäffer, C., Naismith, J. H. (2008b). Purification, crystallization and preliminary crystallographic analysis of WsaF, an essential rhamnosyltransferase from Geobacillus stearothermophilus. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 64, 1163-1165