Evidence for a carbohydrate-binding module (CBM) of Tannerella forsythia NanH sialidase, key to interactions at the host-pathogen interface.

Bacterial sialidases cleave terminal sialic acid from a variety of host glycoproteins, and contribute to survival and growth of many human-dwelling bacterial species, including various pathogens. Tannerella forsythia , an oral, Gram-negative, fastidious anaerobe, is a key organism in periodontal disease and possesses a dedicated sialic acid utilisation and scavenging ( nan ) operon, including NanH sialidase. Here, we describe biochemical characterisation of recombinant NanH, including its action on host-relevant sialoglycans such as sialyl Lewis A and sialyl Lewis X (SLeA/X ), and on human cell-attached sialic acids directly, uncovering that it is a highly active broad specificity sialidase. Furthermore, the N-terminal domain of NanH was hypothesised and proved to be capable of binding to a range of sialoglycans and non-sialylated derivatives with K d in the micromolar range, as determined by steady-state tryptophan fluorescence spectroscopy, but it has no catalytic activity in isolation from the active site. We consider this domain to represent the founding member of a novel subfamily of carbohydrate-binding module (CBM), involved in glycosidase-ligand binding. In addition, we created a catalytically inactive version of the NanH enzyme (FRIP → YMAP) that retained its ability to bind sialic acid-containing ligands and revealed for the first time that binding activity of a CBM is enhanced by association with the catalytic domain. Finally, we investigated the importance of Lewis-type sialoglycans on T. forsythia -host interactions, showing that nanomolar amounts of SLeA/X were capable of reducing invasion of oral epithelial cells by T. forsythia , suggesting that these are key ligands for bacterial-cellular interactions during periodontal disease.