An Orthologue of Bacteroides fragilis NanH Is the Principal Sialidase in Tannerella forsythia

Abstract

ABSTRACT Sialidase activity is a putative virulence factor of the anaerobic periodontal pathogen Tannerella forsythia , but it is uncertain which genes encode this activity. Characterization of a putative sialidase, SiaHI, by others, indicated that this protein alone may not be responsible for all of the sialidase activity. We describe a second sialidase in T. forsythia (TF0035), an orthologue of Bacteroides fragilis NanH, and its expression in Escherichia coli . Sialidase activity of the expressed NanH was confirmed by using 2′-(4-methylumbelliferyl)-α- d - N -acetylneuraminic acid as a substrate. Biochemical characterization of the recombinant T. forsythia NanH indicated that it was active over a broad pH range, with optimum activity at pH 5.5. This enzyme has high affinity for 2′-(4-methylumbelliferyl)-α- d - N -acetylneuraminic acid ( K m of 32.9 ± 10.3 μM) and rapidly releases 4-methylumbelliferone ( V max of 170.8 ± 11.8 nmol of 4-methylumbelliferone min −1 mg of protein −1 ). E. coli lysates containing recombinant T. forsythia NanH cleave sialic acid from a range of substrates, with a preference for α2-3 glycosidic linkages. The genes adjacent to nanH encode proteins apparently involved in the metabolism of sialic acid, indicating that the NanH sialidase is likely to be involved in nutrient acquisition.