ASGR-mediated KC capture of serotype-7F and -14 pneumococci. (A) Dose-dependent blocking of mASGR-mediated TH8707F adherence by free CPS7F, galactose, or GalNAc. n = 3. (B) Alignment of the C-type lectin domains of human and mouse ASGR1. (C) Same as A except using hASGR-transfected CHO cells and serotype-14 pneumococci. n = 3. (D) Same as C except using serotype-7F pneumococci. n = 3. (E) Impact of mASGR on adherence to CHO cells by natural pneumococcal strains with terminal galactose in the CPSs. Bacterial adherence was quantified as in Fig. 7 D. n = 3. (F) Same as E except using hASGR-transfected cells. n = 3. (G) The disaccharide structures of endogenous ASGR ligands and the known terminal galactose-containing capsules in S. pneumoniae. Only CPS14 has the identical signature with the native ligand core structure Galβ1,4GlcNAc. (H) Effect of centrifugation on receptor-mediated bacterial adhesion to host cells. Isogenic serotype-14 or -19F pneumococci were suspended in F-12K medium to a density of 106 CFU/ml; 50 μl of bacterial suspension was added to the monolayers of ASGR-expressing CHO cells in 96-well plates; mild centrifugation (500 g for 5 min) was applied immediately after bacterial suspensions were added to cell monolayers to mimic the sheer force that blood-borne bacteria experience in the blood circulation. n = 3. All data were representative results from two to three independent experiments. Ordinary one-way ANOVA with Tukey’s multiple comparisons test (A, C, and D), two-way ANOVA with Sidak’s multiple comparisons test (E, F, and H), *, P < 0.05, ***, P < 0.001, ****, P < 0.0001.
