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.