Characterization of an α-l-fucosidase from the periodontal pathogen Tannerella forsythia

Abstract

The periodontal pathogen Tannerella forsythia expresses several glycosidases which are linked to specific growth requirements and are involved in the invasion of host tissues. α-l-Fucosyl residues are exposed on various host glycoconjugates and, thus, the α-l-fucosidases predicted in the T. forsythia ATCC 43037 genome could potentially serve roles in host-pathogen interactions. We describe the molecular cloning and characterization of the putative fucosidase TfFuc1 (encoded by the bfo_2737 = Tffuc1 gene), previously reported to be present in an outer membrane preparation. In terms of sequence, this 51-kDa protein is a member of the glycosyl hydrolase family GH29. Using an artificial substrate, p-nitrophenyl-α-fucose (KM 670 μM), the enzyme was determined to have a pH optimum of 9.0 and to be competitively inhibited by fucose and deoxyfuconojirimycin. TfFuc1 was shown here to be a unique α(1,2)-fucosidase that also possesses α(1,6) specificity on small unbranched substrates. It is active on mucin after sialidase-catalyzed removal of terminal sialic acid residues and also removes fucose from blood group H. Following knock-out of the Tffuc1 gene and analyzing biofilm formation and cell invasion/adhesion of the mutant in comparison to the wild-type, it is most likely that the enzyme does not act extracellularly. Biochemically interesting as the first fucosidase in T. forsythia to be characterized, the biological role of TfFuc1 may well be in the metabolism of short oligosaccharides in the periplasm, thereby indirectly contributing to the virulence of this organism. TfFuc1 is the first glycosyl hydrolase in the GH29 family reported to be a specific α(1,2)-fucosidase.

Keywords: 2) fucosidase; 4-nitrophenyl-α-l-fucopyranoside; Amp, ampicillin; BHI, brain heart infusion medium; CBB, Coomassie brilliant blue G 250; DFJ, deoxyfuconojirimycin; Erm, erythromycin; FDH, fucose dehydrogenase; HPAEC, high-performance anion-exchange chromatography with pulsed amperometric detection; LC-ESI-MS, liquid chromatography-electrospray ionisation-mass spectrometry; NAM, N-acetylmuramic acid; PBS, phosphate-buffered saline; SDS-PAGE, sodium dodecylsulphate polyacrylamide gel electrophoresis; T. forsythia, Tannerella forsythia ATCC 43037; TfFuc1, T. forsythia ATCC 43037 fucosidase-1 encoded by the bfo_2737 gene, equally Tffuc1; WT, wild-type bacterium.; enzyme activity; enzyme specificity; oral pathogen; pNP-fucose; periodontitis; rTfFuc-1, recombinant TfFuc1 enzyme; tannerella forsythia; α(1.

Figure 1.

SDS PAGE (A) and Western immunoblot (B) of total cell extracts from T. forsythia WT (lane 2) and ΔTffuc1 strains (lane 3) and of the His₆-tagged rTfFuc1 as purified from E. coli (lane 4), used for activity studies and to raise a polyclonal anti-TfFuc1 antiserum. Western immunoblotting using the anti-TfFuc1 antiserum recognized the protein (∼51 kDa) specifically in the WT strain (lane 2) and indicated absence of the protein in the ΔTffuc1 strain (lane 3), proving that the enzyme was effectively knocked-out. In the preparation of rTfFuc 1 (B, lane 4), the polyclonal antiserum recognizes also minor contaminating E. coli proteins not visible on the SDS-PAGE gel (A, lane 4). Mm; PageRuler Plus prestained protein ladder (Thermo Scientific).

Figure 2.

pH profile of rTfFuc1 using 4-nitrophenyl-α-l-fucopyranoside (pNP-fucose) as a substrate. Activity was measured as the increase in Abs₄₀₅ due to the released 4-nitrophenol product. Citrate/phosphate buffer (0.1 M) was used to assay the pH range from 3–8, 50 mM glycine buffer was used for the pH range from 8.0–10.25.

Figure 3.

Fucosylated substrates used in this study. The structures are depicted according to the symbolic nomenclature of the Consortium for Functional Glycomics (http://www.functionalglycomics.org/static/consortium/Nomenclatures.html).

Figure 4.

rTfFuc1 activity on standard fucosylated substrates after overnight incubation as determined by HPAEC. Blue lines represent samples which were incubated in absence of rTfFuc1 (substrate standard) and red lines represent samples incubated in the presence of rTfFuc1. Cleavage of the substrates was determined by the appearance of a fucose peak, as determined by the retention time of the standard monosaccharide.

Figure 5.

Cleavage of natural α(1,2) fucosylated glycans by rTfFuc1. Cleavage of fucose from a large N-glycan substrate was monitored by MALDI-TOF MS spectra after overnight incubation; the conversion of the m/z 1703 glycan (GalF) to one of m/z 1557 (Δm/z 146) is indicative of the loss of fucose. The structures of the substrate and product are depicted according to the symbolic nomenclature of the Consortium for Functional Glycomics.

Figure 6.

rTfFuc1 was incubated with mucin from bovine submaxillary glands and the release of fucose was measured with the K-FUCOSE kit. When incubations were performed in conjunction with the rNanH sialidase, a slow steady increase in the Abs₃₄₀ was observed. The activity was calculated over a period of 300 s where the data points fitted a linear regression with an r² of 0.98. No activity could be detected when rTfFuc1 was incubated alone with the mucin. The ΔAbs₃₄₀ lead to an irregular data set with a very low r² value of 0.4.

Figure 7.

Presence of TfFuc1 in cell fractions of T. forsythia WT. (A) SDS-PAGE analysis of the outer membrane fraction (OM) (1), membrane fraction (2) and non-membrane associated fraction (3) showed good separation between the fractions, as the S‑layer bands were very prominent in the OM but not in the membrane and non-membrane associated fractions. Protein loaded was 20 μg of the OM and non-membrane associated fractions and 400 μg of the membrane fraction. Protein visualization was by CBB. (B) Western immunoblot using anti-TfFuc1 antiserum showed the TfFuc1 fucosidase in the non-membrane associated fraction comprising both the cytoplasmic and periplasmic content. Mm; PageRuler Plus prestained protein ladder (Thermo Scientific).