Periodontal pathogens harvest sugar from human sialylated proteins, but which ones?

Objectives: Sialic acids are a group of carbohydrates, found extensively in eukaryotic glycoproteins. Periodontal pathogens possess sialidase enzymes which grant the ability to cleave sialic acid from human sialoglycoproteins, serving a variety of functions for pathogens including nutrient acquisition, biofilm formation, immunoregulation, and host cell association. This work aims to identify the sialoglycoproteins targeted by the important red complex pathogen Tannerella forsythia, and the potential use of novel sialidase inhibitors to prevent loss of sialic acid from sialoglycoproteins.
Methods: Labelling cell surface sialoglycoproteins of oral epithelial cell lines was achieved by aniline oxidation of surface sialic acids followed by biotinylation and visualisation by Streptavidin-Texas-Red staining by fluorescent microscopy. Purification of biotinylated sialoglycoproteins with streptavidin agarose beads allowed analysis of the sialoglycoproteome by SDS-PAGE. The effect on the sialoglycoproteome to sialidase from Tannerella forsythia in the presence or absence of sialidase inhibitors was observed.
A fluorescence based assay was used to assess the efficacy of potential inhibitors for pathogen sialidases. Sialidase activity was quantified by release of fluorescent methylumbelliferone from a methyumbelliferyl-sialic acid conjugate by T. forsythia sialidase, in the presence and absence of inhibitors.
Results: Fluorescence microscopy revealed that sialic acids were abundant on surfaces of oral epithelial cells, and treatment with T. forsythia sialidase drastically reduced the level of sialic acid. Extraction of these membrane sialoglycoproteins is ongoing. Novel inhibitors were capable of significantly reducing T. forsythia’s sialidase activity, with some decreasing activity by ~80% at 1mM.
Conclusions: Analysis of the sialoglycoproteome of oral cell lines is ongoing. A range of inhibitors tested proved successful in reducing T. forsythia’s sialidase activity. These results highlight the potential for development of sialidase inhibitors for periodontitis therapy.