Antigenic organization of the N-terminal part of the polymorphic outer membrane proteins 90, 91A, and 91B of Chlamydophila abortus

Abstract

A series of overlapping recombinant antigens, 61 to 74 residues in length, representing polymorphic outer membrane protein 90 (POMP90) of Chlamydophila abortus and two recombinant peptides spanning gene fragment p91Bf99 of POMP91B were assessed by immunoblotting to determine the antigen-binding sites of 20 monoclonal antibodies to POMP90, -91A, and -91B. The epitopes were further restricted by scanning 52 overlapping synthetic 12-mer peptides representing the N-terminal part of POMP90, and the 12-mer epitopes were then analyzed by using hexapeptides to the resolution of a single amino acid. Ten epitopes were defined: 1, TSEEFQVKETSSGT; 2, SGAIYTCEGNVCISYAGKDSPL; 3, SLVFHKNCSTAE; 4, AIYADKLTIVSGGPTLFS; 5, SPKGGAISIKDS; 6, ITFDGNKIIKTS; 7, LRAKDGFGIFFY; 7a, DGFGIF; 7b, GIFFYD; 8, IFFYDPITGGGS; 8a, FFYDPIT; 9, GKIVFSGE; and 10, DLGTTL. The 20-mer peptide LRAKDGFGIFFYDPITGGGS was a major epitope that was recognized by seven antibodies. Epitopes 7 to 10 were conserved in reference strains of the former species C. psittaci, whereas the strong antigenic peptides FYDPIT and IVFSGE were conserved among members of the genus CHLAMYDOPHILA: Epitopes 3 to 8 were located within the best-scoring beta-helical wrap (residues 148 to 293) predicted for POMP91B by the program BETAWRAP. Other studies have suggested an association of the POMPs with type V secretory autotransporter proteins. The results presented in this study provide some evidence for a passenger domain that is folded as a beta-helix pyramid with compact antigenic organization.

FIG. 1.

Binding patterns of MAbs against recombinant protein fragments representing POMP90, expressed as GST fusion proteins (see Table 2). Purified fragments rOMP90-1 to -7 and rOMP90-9 to -11 were subjected to SDS-12% PAGE and immunoblotted with MAbs FC1A2, 73/04, G11, 84/019, and 73/0040, with affinity-purified antibodies to POMP105kDa, and with a pool of postabortion ewe sera.

FIG. 2.

Specificity of MAb JA6C7 for POMP91B. Recombinant fragments expressed as GST fusion proteins covering residues 17 to 87 (rOMP90-1, lane 2) and 72 to 137 (rOMP90-2, lane 1) of POMP90 and residues 44 to 89 of POMP91B (p91Bf99a, lane 3) and the empty-control vector (GST, lane 4) were subjected to SDS-12% PAGE and immunoblotted with MAb JA6C7.

FIG. 3.

Binding of MAbs DA4E8, 73/0200 73/0248, and 73/053 to synthetic hexapeptides offset by one amino acid and spanning residues 281 to 300 of POMP90. Reactivity of the MAbs with the peptides is expressed as the net A₄₀₅.

FIG. 4.

Seroreactivity of synthetic peptides. Fifty-two dodecapeptides spanning the N-terminal part of POMP90 were tested with six serum pools from flocks with ovine enzootic abortion and five individual serum samples from SPF lambs infected with C. pecorum. The data for the six most strongly reacting peptides with A₄₀₅/A_background values of ≥1.50 are shown as the mean values plus the standard deviation for the six serum pools (dark bars) and the five C. pecorum serum samples (open bars).

FIG. 5.

Predicted best beta-helix fold (wrap) in POMP90, -91A, and -91B with the program BETAWRAP. The positions of the epitopes, as determined by epitope mapping, are shown in bold and underlined. Each line represents one rung. A single rung has three strands (B1 to -3) and three turns (T1 to -3). The start positions of strands B1 and B2 are shown.

FIG. 6.

Schematic 3D drawing of the epitope map of the N-terminal part of POMP91B containing a beta-helical domain with six rungs. Five rungs have been placed in accordance with the predicted positions of strands B1 and B2 in each rung calculated by BETAWRAP (shown in Fig. 5). The positions of the epitopes were determined by peptide mapping (Table 2). The parts left and right of the beta-helical domain are not to scale. B1, B2, and B3, beta strands; L, leader peptide.