Two complementary α-fucosidases from Streptococcus pneumoniae promote complete degradation of host-derived carbohydrate antigens

Abstract

An important aspect of the interaction between the opportunistic bacterial pathogen Streptococcus pneumoniae and its human host is its ability to harvest host glycans. The pneumococcus can degrade a variety of complex glycans, including N- and O-linked glycans, glycosaminoglycans, and carbohydrate antigens, an ability that is tightly linked to the virulence of S. pneumoniae Although S. pneumoniae is known to use a sophisticated enzyme machinery to attack the human glycome, how it copes with fucosylated glycans, which are primarily histo-blood group antigens, is largely unknown. Here, we identified two pneumococcal enzymes, SpGH29^C and SpGH95^C, that target α-(1→3/4) and α-(1→2) fucosidic linkages, respectively. X-ray crystallography studies combined with functional assays revealed that SpGH29^C is specific for the Lewis^A and Lewis^X antigen motifs and that SpGH95^C is specific for the H(O)-antigen motif. Together, these enzymes could defucosylate Lewis^Y and Lewis^B antigens in a complementary fashion. In vitro reconstruction of glycan degradation cascades disclosed that the individual or combined activities of these enzymes expose the underlying glycan structure, promoting the complete deconstruction of a glycan that would otherwise be resistant to pneumococcal enzymes. These experiments expand our understanding of the extensive capacity of S. pneumoniae to process host glycans and the likely roles of α-fucosidases in this. Overall, given the importance of enzymes that initiate glycan breakdown in pneumococcal virulence, such as the neuraminidase NanA and the mannosidase SpGH92, we anticipate that the α-fucosidases identified here will be important factors in developing more refined models of the S. pneumoniae-host interaction.

Keywords: Streptococcus; X-ray crystallography; glycoside hydrolase; host-pathogen interaction; structure-function.

Figure 1.

Overall structure of SpGH29^C. The X-ray crystal structure of SpGH29^C is represented as a cartoon colored from blue (N terminus (Nter)) to red (C terminus (Cter)). Both catalytic residues and a Bis-Tris molecule observed to be bound in the active site are shown as gray sticks. The numbering of helices and β-strands comprising the (α/β)₈ catalytic module is indicated. The strand numbering of the ancillary module is also indicated.

Figure 2.

The SpGH29^CT D171N/E215Q catalytic pocket in complex with histo-blood group antigens. A, structure of SpGH29T D171N/E215Q (green sticks) in complex with Lewis^A antigen. The insets focus on the +2′ galactose-binding subsite (top right inset), the −1 fucose-binding subsite (bottom right inset), and +1* GlcNAc pseudo-subsite (left inset). B, structural overlay of BiAfcB D172A/E217A (PDB code 3EUT; orange sticks) in complex with the Lewis^A antigen (gray lines) with the Lewis^A antigen from the complex with SpGH29T D171N/E215Q. The active-site side chains of SpGH29T D171N/E215Q were omitted as they are completely conserved with those of BiAfcB. SpGH29T residue numbering is shown in black, and that of BiAfcB is shown in gray. C and D, SpGH29T D171N/E215Q in complex with the Lewis^X and Lewis^Y antigens, respectively. Fucose, galactose, and GlcNAc are shown in red, yellow, and blue sticks, respectively. The water molecule (wat) is represented by a purple sphere. Dashed lines denote hydrogen bonds. Subsites are indicated in red.

Figure 3.

Example of sequential degradation of a human glycan by pneumococcal GHs. A, the substrate type IV H-tetrasaccharide was incubated with enzyme(s) overnight, and the products were labeled and visualized by fluorophore-assisted carbohydrate electrophoresis. ± above the gel indicates the presence/absence of substrate or enzyme. B, schematic depiction of the sequential breakdown of type IV H-tetrasaccharide by pneumococcal GHs. GHs are indicated in bold next to the arrow for the reaction they catalyze.

Figure 4.

Schematic depiction of the sequential degradation of histo-blood group antigens by pneumococcal GHs. GHs are indicated in bold next to the arrow for the reaction they catalyze. A, degradation of Lewis^Y can be initiated either by SpGH29^C, which yields the type II H-trisaccharide (H-Tri), or by SpGH95^C, which yields Lewis^X. The complementary α-fucosidase then acts to produce N-acetyllactosamine (LacNAc), which is cleaved into its constituent monosaccharides by BgaA. Sialyl-Lewis^X must be desialylated by NanA prior to SpGH29^C activity. B, degradation of Lewis^B can be initiated either by SpGH29^C, which yields the type I H-trisaccharide, or by SpGH95^C, which yields Lewis^A. The complementary α-fucosidase then acts to produce lacto-N-biose, which is cleaved into its constituent monosaccharides by BgaC. Sialyl-Lewis^A must be desialylated by NanA prior to SpGH29^C activity. See Fig. S3 for experimental validation for the sequential depolymerization of each of the boxed species in this figure.

Figure 5.

Cellular localization of SpGH29^C. A and B, activity of different cellular fractions of WT TIGR4 (A) and ΔspGH29^C (B) against Lewis^X as detected by fluorophore-assisted carbohydrate electrophoresis. The activities of recombinant SpGH29^C and BgaA against Lewis^X are shown as controls, and fucose is shown as a standard. C, background labeling of the different cellular fractions in the absence of Lewis^X. Lewis^X and Lewis^X treated with total soluble protein are shown for comparison. Le^X, Lewis^X; EF, extracellular fraction; CF, cytoplasmic fraction; MF, membrane fraction. The 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS) lane indicates background labeling due to the fluorophore alone. Due to the background labeling of the cell wall–associated fraction, SpGH29^C activity can be observed as a disappearance of Lewis^X rather than an appearance of fucose.

Figure 6.

Schematic depiction of the sequential degradation of TFLNH by pneumococcal GHs. GHs are indicated in bold next to the arrow for the reaction they catalyze; numbers in green refer to the gel lane in Fig. S4. SpGH95^C and SpGH29^C are required to remove the capping fucose residues from TFLNH and allow access to the oligosaccharide by other GHs. Treatment of TFLNH with SpGH29^C results in removal of the α-(1→3)– and α-(1→4)–linked fucose units and allows BgaA and GH20C to degrade the arm proximal to the reducing end; however, without SpGH95^C, the distal arm cannot be degraded. Treatment of TFLNH with SpGH95^C results in partial removal of the α-(1→2)–linked fucose unit and a difucosylated oligosaccharide that cannot be acted upon by other GHs (except SpGH29^C). If the α-(1→3)– and α-(1→4)–linked fucose units are removed by SpGH29^C first, SpGH95^C is able to fully remove the α-(1→2)–linked fucose unit from the distal arm to produce lacto-N-hexaose. This hexasaccharide can then be fully degraded into galactose and glucose by the combined actions of BgaA, BgaC, and GH20C. See Fig. S4 for experimental validation of this pathway.