Whilst certain residue substitutions, e.g. CEACAM1 crystal structure, and observe that all of the above residues form an exposed continuous binding region around the N-domain. Astragaloside II Examination of the model also suggested that substitution of two of these residues 34 and 89 could affect the accessibility of Ile-91 for ligand binding. By introducing selected mutations at the positions 91, 34 and 89, we confirmed the primary importance of Ile-91 in all bacterial binding to CEACAM1 despite the inter- and intraspecies structural differences between the bacterial CEACAM-binding ligands. The studies further indicated that this efficiency of binding was significantly enhanced for specific strains by mutations such as Y34F and Q89N, which also altered the hierarchy of Nm versus Hi strain binding. These studies imply that distinct polymorphisms in human epithelial CEACAMs have the potential to decrease or increase the risk of infection by the receptor-targeting pathogens. Introduction The bacterial pathogens (Nm) and (Hi) are frequently found in the nasopharynx of a substantial proportion of the healthy population but are capable of causing serious infections in susceptible individuals (Turk, 1984; Foxwell and enteric pathogens and (Leusch and P5 proteins of Hi (Chen and Gotschlich, 1996; Virji contain multiple copies of genes that encode conserved domains which form -barrel structures in bacterial membranes and variable domains that form surface uncovered loops. In spite of the surface diversity afforded by the hyper-variable domains of the loops, the majority of the Opa proteins are capable of targeting CEACAMs (Virji binding to CEACAM1 constructs Opa-expressing phenotypes of two Nm strains (C751 and MC58) were used to investigate their binding to the altered NA1B-Fc receptors. Three C751 derivatives expressing distinct Opa proteins (OpaA, OpaB and OpaD) and the MC58 derivative expressing an Opa protein designated OpaX (Virji isolates expressing distinct Opa proteins. Bacterial lysates were dotted on to nitrocellulose and overlaid with NA1B-Fc constructs as indicated. Binding relative to the Astragaloside II native receptor was determined by densitometric analysis of immno-blots using NIH Scion Image programme. One hundred per cent binding level is usually Astragaloside II indicated by the horizontal line allowing comparison to native receptor binding. Mean values and SE of 3 replicates are shown in each case, except Q89A (= 1) for OpaA, B and X. However, alanine substitution at all three positions confirm previous observations (Virji isolate C751OpaB to cell-expressed receptors: competition between cell-expressed and soluble receptors. Bacterial binding to cell-expressed CC1 or Q89N construct was detected using anti-Nm antisera and TRITC-conjugated secondary antibodies. The interactions of bacteria with the cell-expressed receptors were investigated in the absence (A, D) or presence of competing soluble receptors. Although binding to the soluble receptor Q89N is much lower than the native CC1 in dot-blots (Fig. 3B), it is relatively high on certain cells (presumably, on those expressing high levels Astragaloside II of the receptor) (D). In competitive experiments, the native CC1-Fc (B, E) and the CC1(Q89N)-Fc (C, F) were preincubated at 10 ug ml?1 with bacteria for 15 min, prior to contamination of target cells. CC1-Fc inhibited bacterial binding to the homologous receptor significantly (B), and almost abrogated binding to cell-expressed Q89N (E); whereas the soluble Q89N was inefficient at inhibiting bacterial binding to CHO-CC1 (C). However, a level of homologous inhibition was apparent when examining the adhesion of bacteria to cells expressing low levels of the receptor (e.g. peppered areas shown in D are less evident in F). binding to CEACAM1 constructs One non-typable (A950002) and two THi strains (Rd and Eagan) were used in receptor overlay experiments as above (Fig. 4). Alanine substitutions at Ile-91 confirmed previous results (Virji strains. Bacterial lysates immobilized on nitrocellulose were overlaid with NA1B-Fc constructs. Binding relative to the native receptor was decided as in legend to Fig. 3. Mean values and SE of three IL10RB to five replicates are shown. (D) Control dotCblots showing the interactions of CC1-binding (Rd) and non-binding (RdCC-) derivatives of the THi strain with the native and Q89N receptors..