Ehrlichia canis gp200 contains dominant species-specific antibody epitopes in terminal acidic domains

Abstract

Species-specific antibody epitopes within several major immunoreactive protein orthologs of Ehrlichia species have recently been identified and molecularly characterized. In this study, dominant B-cell epitopes within the acidic (pI 5.35) ankyrin repeat-containing 200-kDa major immunoreactive protein (gp200) of Ehrlichia canis were defined. The E. canis gp200 gene (4,263 bp; 1,421 amino acids) was cloned and expressed as four (N-terminal, 1,107 bp; N-internal, 910 bp; C-internal, 1,000 bp; and C-terminal, 1,280 bp) overlapping recombinant proteins. The N-terminal, C-internal, and C-terminal polypeptides (369, 332, and 426 amino acids, respectively) were strongly recognized by antibody, and the major epitope(s) in these polypeptides was mapped to four polypeptide regions (40 to 70 amino acids). Smaller overlapping recombinant polypeptides (14 to 15 amino acids) spanning these regions identified five strongly immunoreactive species-specific epitopes that exhibited conformational dependence. The majority of the epitopes (four) were located in two strongly acidic (pI 4 to 4.9) domains in the distal N- and C-terminal regions of the protein flanking the centralized ankyrin domain-containing region. The amino acid content of the epitope-containing domains included a high proportion of strongly acidic amino acids (glutamate and aspartate), and these domains appear to have important biophysical properties that influence the antibody response to gp200.

FIG. 1.

Schematic of E. canis gp200 showing predicted isoelectric points (pIs) of acidic terminal domains and the slightly basic central ankyrin repeat (22 shaded boxes)-containing region. The cloned recombinant expressed regions (Nt, Ni, Ci, and Ct) are shown, and the approximate locations of mapped epitopes are designated by arrows. aa, amino acids.

FIG. 2.

(A) SDS-PAGE and total protein stain showing four E. coli-expressed overlapping E. canis gp200 recombinant proteins representing 99% of the open reading frame. Lane 1, Nt; lane 2, Ni; lane 3, Ci; lane 4, Ct. Molecular masses (kilodaltons) are shown on the left. (B) Corresponding Western immunoblot probed with polyclonal dog anti-E. canis serum (2995). Lanes and numbers at left are as defined for panel A. Negative controls (ctrl) are recombinant E. chaffeensis Dsb (22 kDa) (A) and recombinant chloramphenicol acetyltransferase (28 kDa) (B).

FIG. 3.

Immunoreactivities of 24 overlapping recombinant polypeptides covering the major immunoreactive regions of the E. canis gp200. Lanes and lines: 1, Nt_8-186; 2, Nt_8-66; 3, Nt_66-100; 4, Nt_29-72; 5, Nt_8-35; 6, Nt_86-186; 7, Ci_650-765; 8, Ci_757-879; 9, Ci_876-1024; 10, Ci_757-817; 11, Ci_808-878; 12, Ci_862-937; 13, Ci_933-985; 14, Ci_979-1024; 15, Ci_876-917; 16, Ci_915-947; 17, Ci_942-985; 18, Ct_1222-1395; 19, Ct_1222-1274; 20, Ct_1292-1340; 21, Ct_1222-1340; 22, Ct_1266-1298; 23, Ct_1332-1371; 24, Ct_1363-1404. Nt, Ci, and Ct represent their locations in the large N-terminal, C-internal, and C-terminal regions of gp200, respectively (subscripts designate the amino acid numbers included in each fragment). (A) Membrane total protein staining. (B) Western immunoblots probed with polyclonal dog anti-E. canis serum. (C) Schematic of E. canis gp200 illustrating the locations of the 24 recombinant fragments and the epitope-containing regions. Solid lines, strongly immunoreactive recombinant gp200 fragments; dotted lines, weakly immunoreactive or nonimmunoreactive recombinant gp200 fragments; hatched boxes, epitope-containing regions; Ctrl, negative-control recombinant thioredoxin; ns, not shown. Molecular masses (kilodaltons) are shown on the right.

FIG. 4.

Immunoreactivities of recombinant and synthetic overlapping 14- and 15-mer peptides (Table 3) spanning the epitope-containing regions of E. canis gp200. Positive control (Ctrl or +), E. chaffeensis p28-19 peptide epitope; lane and x-axis numbers correspond to the peptide numbers in Table 3. (A) Membrane total protein staining. (B) Western immunoblot probed with polyclonal dog anti-E. canis serum. (C) Immunoreactivities of the recombinant gp200 peptides and the corresponding synthetic gp200 peptides expressed as the average optical densities at 650 nm of quadruplet samples as determined by ELISA. The recombinant E. canis gp200 regions corresponding to the overlapping peptides are labeled under the x axis. Molecular masses (kilodaltons) are shown on the left.

FIG. 5.

Comparison of antibody reactivities as determined by ELISA between wild type (WT) and single serine-to-alanine mutant recombinant E. canis gp200 peptides 2 and 20 containing dominant epitopes. OD, optical density.