Structural Insights into Polymorphic ABO Glycan Binding by Helicobacter pylori

Abstract

The Helicobacter pylori adhesin BabA binds mucosal ABO/Le(b) blood group (bg) carbohydrates. BabA facilitates bacterial attachment to gastric surfaces, increasing strain virulence and forming a recognized risk factor for peptic ulcers and gastric cancer. High sequence variation causes BabA functional diversity, but the underlying structural-molecular determinants are unknown. We generated X-ray structures of representative BabA isoforms that reveal a polymorphic, three-pronged Le(b) binding site. Two diversity loops, DL1 and DL2, provide adaptive control to binding affinity, notably ABO versus O bg preference. H. pylori strains can switch bg preference with single DL1 amino acid substitutions, and can coexpress functionally divergent BabA isoforms. The anchor point for receptor binding is the embrace of an ABO fucose residue by a disulfide-clasped loop, which is inactivated by reduction. Treatment with the redox-active pharmaceutic N-acetylcysteine lowers gastric mucosal neutrophil infiltration in H. pylori-infected Le(b)-expressing mice, providing perspectives on possible H. pylori eradication therapies.

Figure 1. BabA^AD Interacts with Lewis b bg Antigens

(A) Schematic of the BabA architecture. Arrows indicate the aa 25–460 BabA adhesin domain fragment (BabA^AD). Abbreviations: CL, cysteine-clasped loops; TM, predicted transmembrane domain; ID, Bab insertion domain (Figures S2 and S3). (B) ITC injection heats (upper) and normalized binding isotherm (lower) of the BabA^AD titrated with Le^b5. (C) SPR sensorgram of full-length BabA (solid and dashed lines show raw and fitted binding curves, respectively, for 500, 250, 125, 62.5, 31.3, and 15.7 nM BabA, from the top down; with a dissociation constant K_d = 3.9E-10 ± 0.9E-10 (M), an association rate constant k_a = 6.1E5 ± 1.4E5 (M⁻¹ s⁻¹), and slow dissociation rate constant, k_d = 2.3E-4 ± 0.8E-4 (s⁻¹). (D) Similar SPR sensorgram of purified BabA^AD binding to a Le^b-coated chip; [BabA] as in (C). (E) Immunoblot detection of BabA from glutaraldehyde(GA) crosslinked H.pylori 17875/Leb bacterial cells; M, monomer, O, BabA oligomer. See also Figure S1.

Figure 2. Crystal Structure of BabA^AD

(A) X-ray structure of strain 17875 BabA^AD (for clarity, Nb-ER19 is not shown, see Figure S1D). Helices and strands are colored red and green, respectively; Cys-bound loops CL1 (Cys106-Cys135), CL2 (Cys189-Cys197), CL3 (Cys277-Cys299), and CL4 (Cys395-Cys423) are colored blue, orange, green, and yellow, respectively. Bab ID: residues 175–255 (Figures S2 and S3). (B) Schematic alignment of Cys-loop topology (vertical marks, colored as in Figures 1A and 1B) in the known or suspected Hop family adhesins. The α-helical ectodomain and β strand domains are colored brown and blue, respectively (sequence lengths not to scale, see Figure S2 for full MSA). (C) Structure of 17875 BabA^AD (colored as in Figure 2) bound to Le^b6 (Table S1). Le^b6 and interacting amino acids (labeled) are shown in stick representation (O, N, and S atoms are colored red, blue, and yellow, respectively). Two binding subsites can be identified: the α1-2 fucose binding pocket (boxed orange) formed by CL2 and T246 in strand S6, and the type 1 chain binding region (boxed magenta) formed by the loop connecting strands S5 and S6 (i.e., DL2, see Figure 3). (D) H-bond network steering the 17875 BabA^AD-Le^b interaction. Side-chain- and main-chain-mediated H-bonds are depicted as green and red arrows, respectively. (E) Schematic of ABO Lewis b bg antigens (see Table S1), with monosaccharides labeled A–G.

Figure 3. Structure of BabA^AD Bound to Lewis b bg H Hexasaccharide

(A) Solvent-accessible surface of BabA^AD, with blue, white, and yellow corresponding to high, medium, and low sequence conservation in multiple sequence alignment of 237 publicly available BabA sequences. Four out of five regions of increased sequence diversity map to the same side of the adhesin: (i) the loop connecting CL1 and H4 (CL1-H4; residues 136–146); (ii and iii) two loops in the Bab ID, e.g., DL1 (Diversification Loop 1; residues 200–210, connecting CL2 and S4) and DL2 (residues 234–242, connecting S5 and S6) (Figures 2D and S3); and (iv) CL3 (residues 279–299). (B) Superimposition of strain 17875 BabA^AD(colored as Figure 2C), with mutant BabA^AD where the strain 17875 insertion domain is replaced by that of strain P437 (blue) or A730 (green). (C) Sequence conservation plot of the BabA insertion domain. Residues that interact with the secretor fucose and core 1 moiety are highlighted by asterisks and squares, respectively. See also Figure S3 and Table S2.

Figure 4. The CL2 Disulfide Bond Is Crucial for High-Affinity Le^b Binding

(A) Normalized radioimmunoassay (RIA) of H. pylori strain 17875/Leb binding to ¹²⁵I-labeled Le^b in presence of increasing concentrations of DTT, added prior to (dotted lines) or coincubated with (solid lines) Le^b. DTT-exposed bacteria resuspended in DTT-free buffer for 2 hr, show recovery of Le^b binding (red line). Data points show mean ± SD, n = 2. (B) RIA Le^b-binding of (left) the J166CL2 mutant (Cys189Ala and Cys197Ala), J166 WT, and J166ΔbabA; and (right) a babA deletion mutant of strain P1 (P1ΔbabA) and this strain conjugated with a shuttle vector expressing WT or CL2 mutant babA from strain 17875. Inlays show α-BabA immunoblots of the corresponding strains. Data points show mean ± SD, n = 2. (C) RIA experiment showing relative Le^b binding of H. pylori clinical isolates preincubated with DTT. (D) SPR sensorgrams of the H. pylori strains with plasmid-based 17875 babA expression CL2 or WT BabA (black or gray response curves, respectively). Three dilutions of bacteria were flushed over immobilized Le^b receptor conjugates, amplification of the CL2 curves are shown in inset. See also Figure S4.

Figure 5. Treatment of H. pylori cells with N-Acetylcysteine Blocks BabA Adherence

(A) FITC-labeled 17875/Leb bacteria binding to human gastric tissue sections. Prior to binding, bacteria were preincubated with 0, 10, or 20 mg/mL NAC (i, ii, and iii, respectively), resulting in 100%, 14%, and less than 5% adherent bacteria, respectively. In panels iv, v, and vi, tissue sections with bound bacteria are treated with 10, 20, or 200 mg/mL NAC, resulting in undetectable, 50%, and over 90% bacterial detachment, respectively. (B) Normalized RIA Le^b-binding of H. pylori strain 17875/Leb when being subjected to increasing concentrations of N-acetylcysteine (0, 10, 20, 30, and 50 mg/mL) for 1 hr at 37°C. Data points show mean ± SD, n = 3. (C and D) H. pylori epithelial adherence and neutrophil recruitment in gastric epithelium of mice treated for 2 weeks with 0 (−) or 40 (+) mg/day NAC in their drinking water. Data points show, per animal, mean bacterial counts (n = 3) per mm of immunostained gastric epithelium (C; Figure S5D), or mean neutrophil counts (n = 3) per mm² of immunohistostained gastric sections (D; Figure S5E). Statistical comparison of the groups produced a Welch-corrected t(9) = 2.29, *p = 0.0475; and t(7) = 7.559, ***p = 0.0001, respectively. Horizontal lines show sample mean ± SD, n = 8 (−NAC) and 9 (+NAC). See also Figure S5.

Figure 6. BabA Binding to bg A and B Glycans

Overlay of the structures of BabA^AD bound to Le^b6 (colored as Figure 3B) or BLe^b7 (Table S1; yellow). Le^b6 and BLe^b7 are shown in stick representation, as are glycan-binding amino acids (shown for BabA^AD Le^b6 complex, and Glu192 and Gln207 for the BLe^b7 complex). Hydrogen bonds present in both the Le^b6 and BLe^b7 interaction or specific to BabA^AD:BLe^b7 (see arrows) are shown in black or yellow dashed lines, respectively. Inset shows detail (rotated up by ∼45°) of the subpocket binding the bg B Gal. Complexes with ALe^b5 or A6-1 are shown in Figure S6D.

Figure 7. Molecular Determinants of BabA bg Preference

(A) Structure of the BabA S831 DL1 grafted hybrid (BabA^AD;DL1-S831; tan, positions 198–199 in magenta) in complex with Le^b6 (magenta), and wild-type 17875 BabA^AD bound to BLe^b7 (light gray, with DL1 region in cyan and positions 198–199 in blue). In the specialist hybrid the replacement of Lys199 with Pro199 results in the inward rotation of residue 198. Replacement of Ser198 of 17875 by Leu198 causes a steric occlusion of the Gal or GalNAc determinants in BLe^b or ALe^b. (B and C) SPR sensorgrams of binding of BabA^AD;DL1-S831 to a Le^b-coated chip in the presence of competing soluble glycans: Le^b5 (B) or ALe^b5 (C), added in a 2-fold dilution series from 5 mM to 1 μM, colored gray to black. (D) RIA of Le^b versus ALe^b binding of a generalist (S831G[D]) and specialist clone (S831S), and the control strains S831 and 17875/Leb. Amino acid sequence ofthe DL1 region (shown starting at Cys197) of the babA allele in the two loci of the corresponding strains. Binding and sequencing data are representative for two independent S831G(D) and S831S clones isolated. (E) H. pylori 17875/Leb demonstrates the generalist phenotype and ability to bind both Alexa 488-labeled Le^b (i) and Alexa 555-labeled ALe^b (ii). Probing of theoriginal sweep population of the specialist H. pylori strain S831 by Alexa 555-labeled ALe^b conjugate demonstrated by fluorescence microscopy the rarepresence of S831 bacterial cells of the generalist phenotype (F) Competition RIA where Le^b binding to a S831G(D) (blue) and S831S (magenta) clone is performed in competition with nonradiolabeled ALe^b. Increasing concentrations of ALe^b titrate out 34.6% of the Le^b binding signal, demonstrating the concomitant expression of a generalist and a specialist babA variant. Data points show mean ± SD, n = 3. See also Figure S7.