Unravelling the multiple functions of the architecturally intricate Streptococcus pneumoniae β-galactosidase, BgaA

Abstract

Bacterial cell-surface proteins play integral roles in host-pathogen interactions. These proteins are often architecturally and functionally sophisticated and yet few studies of such proteins involved in host-pathogen interactions have defined the domains or modules required for specific functions. Streptococcus pneumoniae (pneumococcus), an opportunistic pathogen that is a leading cause of community acquired pneumonia, otitis media and bacteremia, is decorated with many complex surface proteins. These include β-galactosidase BgaA, which is specific for terminal galactose residues β-1-4 linked to glucose or N-acetylglucosamine and known to play a role in pneumococcal growth, resistance to opsonophagocytic killing, and adherence. This study defines the domains and modules of BgaA that are required for these distinct contributions to pneumococcal pathogenesis. Inhibitors of β-galactosidase activity reduced pneumococcal growth and increased opsonophagocytic killing in a BgaA dependent manner, indicating these functions require BgaA enzymatic activity. In contrast, inhibitors increased pneumococcal adherence suggesting that BgaA bound a substrate of the enzyme through a distinct module or domain. Extensive biochemical, structural and cell based studies revealed two newly identified non-enzymatic carbohydrate-binding modules (CBMs) mediate adherence to the host cell surface displayed lactose or N-acetyllactosamine. This finding is important to pneumococcal biology as it is the first adhesin-carbohydrate receptor pair identified, supporting the widely held belief that initial pneumococcal attachment is to a glycoconjugate. Perhaps more importantly, this is the first demonstration that a CBM within a carbohydrate-active enzyme can mediate adherence to host cells and thus this study identifies a new class of carbohydrate-binding adhesins and extends the paradigm of CBM function. As other bacterial species express surface-associated carbohydrate-active enzymes containing CBMs these findings have broad implications for bacterial adherence. Together, these data illustrate that comprehending the architectural sophistication of surface-attached proteins can increase our understanding of the different mechanisms by which these proteins can contribute to bacterial pathogenesis.

Figure 1. The structural features of BgaA from S. pneumoniae.

(A) The architecture of BgaA showing the 17 modules/domains defined on the basis of fold recognition using the Phyre2 server . The 7 different modules/domains are labeled with Arabic numbers: 1, sequence similarity to GH2-associated Ig-like; 2, sequence similarity to GH2 (β/α)₈-barrel; 3, fold similarity to PDB ID 2LY7 (>98% confidence); 4, fold similarity to a fragment of a bacterial invasin (95% confidence); 5, fold similarity to bacterial Ig-like modules (98% confidence); 6, predicted β-sandwich fold similar to that of family 32 CBMs (99.5% confidence); 7, fold similarity to pneumococcal G5 modules (>98% confidence). The LPXTG cell wall anchoring motif is shown. The modules/domains that are the focus of this study are labeled beneath the schematic with Roman numerals. Amino acid numbering for the module/domain boundaries is given above the schematic. (B) Cartoon representation of the structure of the catalytic region comprising domains I–V (colored sequentially as gray, yellow, purple, blue, and orange). The bound LacNAc molecule is shown as green sticks and the surface of the active site in transparent gray. (C) Specific interactions of the BgaA active site with LacNAc (green). Water molecules are shown as red spheres and hydrogen bonds as dashed lines. (D) Overlap of the BgaA active site (purple stick representation for side chains, green sticks for LacNAc, and red spheres for waters) with the active site of E. coli LacZ in complex with lactose (tan stick representation for side chains, orange sticks for lactose, and blue spheres for waters, green sphere for Mg²⁺, and purple sphere for Na⁺; PDB ID 1JYN).

Figure 2. Characterization of BgaA inhibitors.

(A) and (D) The chemical structures of GIF and GNJ, respectively. (B) and (E) Plots of the apparent Km against GIF and GNJ concentration, respectively. Solid lines represent the best fits from linear regression analysis. (C) and (F) Dixon plots of the data shown in panels (B) and (E) for GIF and GNJ, respectively. (G) Specific interactions of the BgaA active site with GIF (yellow sticks) and GNJ (orange sticks) with the protein from the GIF complex shown. Water molecules are shown as red spheres, ethylene glycol molecules as brown sticks, and hydrogen bonds as dashed lines. (H) Divergent stereo view of an overlap of the BgaA LacNAc complex (purple stick representation for side chains, green sticks for LacNAc, and red spheres for waters) with the BgaA GIF complex (blue stick representation for side chains, yellow sticks for LacNAc, brown sticks for a bound ethylene glycol, and black spheres for waters). The surface of the pocket accommodating the O6 of the GlcNAc residue is shown as transparent blue.

Figure 3. Inhibition of S. pneumoniae BgaA.

(A) Growth curves of S. pneumoniae (TIGR4) performed using a semi-defined medium supplemented with bovine asialofetuin. Circles represent growth of the TIGR4 strain, triangles the growth of TIGR4 strain in the presence of 1000 nM GIF, and inverted triangles the growth of the ΔbgaA strain. Also shown as squares is the growth of the TIGR4 strain in the absence of a carbon source. Error bars represent the standard deviation of triplicate experiments run in parallel. The experiment was performed three times with highly similar results. (B) Inhibition of growth on asialofetuin as a function of GIF concentration. Results represent the mean measurements of three independent experiments where culture densities were taken at 6 hr of culture growth. The error bars represent the standard deviations of the independent measurements. (C) Survival of S. pneumoniae TIGR4 in neutrophil killing assays, showing comparisons of wild-type (filled bars) and ΔbgaA strain (open bars) in the presence and absence of GIF. Asterisks above sample bars represent statistical comparison of that sample with the reference, which is the TIGR4 strain with no inhibitor. Data are mean values compiled from two independent experiments performed in duplicate ± standard deviation. The ΔbgaA samples with inhibitors were compared with ΔbgaA in the absence of inhibitors and were found to have p values>0.1 and thus were not significantly different. (D) Addition of GIF (25–2500 nM) significantly increases adherence of S. pneumoniae R6 to D562 cells. Data are the mean ± SD of four independent experiments performed in triplicate. Asterisks above sample bars represent statistical comparison of R6 and R6 + GIF. (E) Adherence of an S. pneumoniae strain expressing enzymatically inactive BgaA (R6BgaAE564R) to D562 cells is significantly higher than the adherence of parental strain (R6). Data are the mean ± SD of three independent experiments performed in triplicate. Asterisks above sample bars represent statistical comparison between R6 and R6BgaAE564R. Statistically significant differences were assessed using a two-tailed Student's t-tests. * p≤0.05, ** p≤0.007, *** p≤0.0007.

Figure 4. The C-terminal region of BgaA is sufficient to facilitate BgaA mediated pneumococcal adherence.

(A) Schematic indicating regions of BgaA deleted to generate BgaAN and BgaAC. Dashed lines indicate regions deleted and numbers indicate amino acid boundaries of the deletions. The signal sequence, shown as the black box, and the Gram positive anchor domain, shown as a grey boxβ, were retained in both constructs for proper localization of expressed proteins. (B) R6BgaAC has significantly higher adherence to NHBE cells as compared to R6ΔbgaA. Adherence of R6BgaAN is not significantly different when compared to R6ΔbgaA. (C) CO6_18BgaAC has significantly higher adherence to NHBE cells as compared to CO6_18ΔbgaA. Adherence of CO6_18BgaAN is not significantly different when compared to CO6_18ΔbgaA. Adherence data are the means ± SD of three independent experiments performed in triplicate. Statistically significant differences were assessed using a two-tailed Student's t-tests. * p≤0.03, ** p≤0.002.

Figure 5. Structures of the CBM71 modules in BgaA.

(A) A cartoon representation of CBM71-1 in complex with LacNAc. The protein is color ramped red to blue from the N-terminus to the C-terminus. A bound calcium atom is shown in magenta. The binding site of the CBM is shown as a grey transparent surface with the bound LacNAc molecule shown as green stick. The electron density for the LacNAc is shown as a blue-mesh F _o-F _c maximum-likelihood/σ_A-weighted map contoured at 3σ (0.33 e⁻/ų). (B) An overlap of the structure of CBM71-2 (purple) with CBM71-1 (grey). The LacNAc molecule bound to CBM71-1 is shown as green sticks. (C) Expanded view of the CBM71 binding site shown in divergent stereo. CBM71-1 is shown in grey with the LacNAc shown as green sticks, residues involved in binding the LacNAc as grey sticks, and the interacting water network as red spheres. Black dashed lines represent potential hydrogen bonds. CBM71-2 is shown in purple; residues conserved with CBM71-2 are shown as purple sticks. (D) An overlap of the CBM71-1 LacNAc complex (grey with LacNAc in green) with the CBM32 from C. perfringens NagJ (blue with bound LacNAc shown as blue sticks).

Figure 6. Recombinant CBMs and LacNAc and/or lactose reduce pneumococcal adherence in a BgaA-dependent manner.

(A) Adherence of S. pneumoniae strain R6 and R6ΔbgaA to NHBE cells in the presence of CBM71-1, CBM71-2 or CBM71-1.2 (250 µM). Asterisks indicate significant differences in adherence in the presence or absence of CBM. (B) Adherence of S. pneumoniae strain C06_18 and C06_18ΔbgaA to NHBE cells in the presence of CBM71-1, CBM71-2 or CBM71-1.2 (250 µM). (C) Adherence of S. pneumoniae strain R6 and R6ΔbgaA to NHBE cells in the presence of LacNAc and lactose (0–10 mM). Asterisks indicate significant differences in adherence in the presence or absence of disaccharide. (D) Adherence of S. pneumoniae strain C06_18 and C06_18ΔbgaA to NHBE cells in the presence of LacNAc and lactose (0–10 mM). As in (C) except using pneumococcal strain C06_18. Adherence assays are mean ± SD of three independent experiments each performed in triplicate. Statistically significant differences were assessed using a two-tailed Student's t-test. * p≤0.04, ** p≤0.007, *** p≤8×10⁻⁴.

Figure 7. CBMs within BgaA directly bind epithelial cells.

(A) Binding of D562 cells to immobilized recombinant CBM71-1. A range of concentrations (2.5–20 µg ml⁻¹) of CBM71-1 were immobilized on the bottom of a 96 well plate. D562 cells pretreated with 0.002 Unit ml⁻¹ C. perfringens sialidase (CpSia) with or without 0.054 µM SpBgaA146-990 (SpBgaA) were allowed to adhere to the plate. Adherence to control wells coated with 1 mg ml⁻¹ of BSA was subtracted from the data (maximum 22 cells per well). Asterisks indicate significant differences in the number of adherent D562 cells following pretreatment with sialidase and sialidase plus SpBgaA146-990. (B) Binding of D562 cells to immobilized recombinant CBM71-2. As in (A) except using CBM71-2. (C) Adherence of S. pneumoniae strain R6 and R6BgaAW1514A,W1864A to D562 cells. Asterisks indicate significant differences in adherence of R6 and R6BgaAW1514A,W1864A. Data are the mean ± SD of three independent experiments each performed in triplicate. Statistically significant differences were assessed using a two-tailed Student's t-test. * p≤0.04, ** p≤2.00×10⁻⁷.

Figure 8. S. gordonii bgaA can restore the β-galactosidase activity and adherence of a S. pneumoniae bgaA mutant.

(A) S. gordonii bgaA can restore the adherence of a S. pneumoniae bgaA mutant. (B) S. gordonii bgaA can restore the β-galactosidase activity of a S. pneumoniae bgaA mutant. Data presented here are mean ± SD of three independent experiments each performed in triplicate. Asterisks indicate statistically significant differences between R6ΔbgaA and both R6ΔbgaA SgbgaA ⁺ and R6 calculated using a two-tailed Student's t-test. * p≤0.002, ** p≤5×10⁻⁶.