An orthologue of Bacteroides fragilis NanH is the principal sialidase in Tannerella forsythia

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)-alpha-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)-alpha-D-N-acetylneuraminic acid (K(m) of 32.9 +/- 10.3 microM) 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 alpha2-3 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.

FIG. 1.

T. forsythia genetic loci containing genes encoding putative sialidases. The genes encoding the putative sialidases in T. forsythia are represented by the black arrows. Surrounding these are genes encoding putative outer membrane proteins (diagonal hatching, panel A only), a transport protein (cross-hatching, panel A only), enzymes (no hatching), or hypothetical proteins of unknown function (vertical hatching). The numbers correspond to gene numbers TF00xx (panel A, TF0030 to TF0038) or TF22xx (panel B, TF2211 to TF2202). The binding sites of the primers used to amplify nanH (A) and siaHI (B) are indicated by vertical lines.

FIG. 2.

Sialidase activity of T. forsythia NanH and SiaHI expressed in E. coli. Strains KCL116 (E. coli pET30; lanes 1 and 2), KCL117 [E. coli BL21(DE3)/pET30::nanH; lanes 3 and 4], and KCL120 [E. coli BL21(DE3)/pET30::siaHI; lanes 5 and 6] were grown in LB until mid-exponential phase and for a further 2 h with (lanes 2, 4, and 6) or without (lanes 1, 3, and 5) induction with 1 mM IPTG. Cells were assayed for sialidase activity in a filter paper spot assay using the fluorogenic substrate 4-MU-NeuNAc (spots in the black boxes above the corresponding lanes). Cell lysates were separated by sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis, and proteins were visualized by Coomassie blue staining.

FIG. 3.

pH optimum of T. forsythia NanH. Strain KCL117 [E. coli BL21(DE3)/pET30::nanH] was grown in LB until mid-exponential phase and for a further 18 h after induction with 1 mM IPTG. Cell lysates were prepared as described in Materials and Methods and assayed using 4-MU-NeuNAc in sodium citrate buffer (pH 3.0 to 6.0), sodium phosphate buffer (pH 6.0 to 8.0), potassium phosphate buffer (pH 6.5 to 7.5), and Tris-HCl (pH 7.5 to 8.9). The release of 4-MU was determined as fluorescence intensity (excitation and emission wavelengths of 380 and 460 nm, respectively). The data shown are the means from at least three independent experiments. Error bars represent ± the standard error of the mean.

FIG. 4.

Schematic representation of the proposed pathway for sialic acid metabolism in T. forsythia highlighting the initiating role of NanH. The removal of sialic acid from the terminus of glycoproteins by NanH allows it to pass through the outer membrane; we propose that this occurs with the help of the proteins encoded by TF0033 and TF0034. The free sialic acid is most likely further transported into the bacterium by the MFS permease encoded by TF0032. TF0030 and TF0031 encode the initial enzymes for sialic acid catabolism, NanA and NanE.