Antigenic sites on porin of Haemophilus influenzae type b: mapping with synthetic peptides and evaluation of structure predictions

Abstract

The major surface-located protein in the outer membrane of Haemophilus influenzae type b (Hib) is porin, molecular mass, 38 kDa, 341 amino acids. To define precisely the molecular reactivities of nine mouse monoclonal antibodies (MAbs) against Hib porin, overlapping hexapeptides corresponding to the entire sequence of porin were synthesized. The epitopes recognized by the MAbs were mapped by enzyme-linked immunosorbent assay to stretches of 6 to 11 amino acids. Antigenic sites between amino acids 112 and 126, 148 and 153, 162 and 172, and 318 and 325 were identified. The antigenic sites between amino acids 162 and 172 and between amino acids 318 and 325 were determined by flow cytometry to be on the bacterial cell surface. Four MAbs, POR.2, POR.3, POR.4, and POR.5, that react with amino acids 162 to 172 were able to discriminate among porins from the three major outer membrane protein subtypes of Hib, i.e., 1H, 2L, and 6U. A model for the topological organization of Hib porin was created by calculating the hydrophobicity, amphiphilicity, and turn propensity in its amino acid sequence. Determination of the molecular reactivities of the anti-Hib porin MAbs provided substantive evidence for the orientation of selected regions of porin in the outer membrane of Hib.

Purification and refolding of recombinant Haemophilus influenzae type b porin produced in Bacillus subtilis

“…Based on the hydrophobicity, amphiphilicity and turn propensity of the amino acid sequence of Hib porin, we generated [6] a secondary structure model of the topological organization of Hib porin. Our model proposed that Hib porin is folded into a [3-barrel with 16 antiparallel strands, eight short periplasmic loops and eight long surface-exposed loops.…”

Topological analysis of the Escherichia coli ferrichrome-iron receptor by using monoclonal antibodies

“…MAbs recognize their determinants with exquisite specificity and are ideal tools with which to deduce topological organization because they bind to short linear sequences or to conformational determinants (31,38,41). We produced and characterized 35 FhuAspecific MAbs, mapped their determinants, and measured their abilities to inhibit the interactions between the native FhuA receptor and its cognate ligands.…”

Lower respiratory tract infection

“…As noted above, the conserved portions of OMPs which are buried within the outer membrane are inaccessible to antibodies in the intact bacte- (93). Immunoassays which reliably detect antibodies to surface epitopes include functional assays (bactericidal and opsonophagocytosis assays), flow cytometry (16,157), adsorption assays (109,110,123,151), elution assays (54,151), and whole-cell radioimmunoprecipitation assays (75). In addition, assays such as ELISAs, immunofluorescence microscopy, and immunoelectron microscopy can detect antibodies to surface-exposed epitopes when performed such that antibodies are allowed to bind to nondenatured whole bacterial cells before fixation.…”