Novel immunoreactive glycoprotein orthologs of Ehrlichia spp.

We previously identified a strongly immunoreactive 43 kDa protein (p43) of Ehrlichia canis. As an immunodiagnostic antigen, the p43 had a 96% accuracy as compared with IFA and provided species-specific diagnosis of E. canis infections. Further investigation has revealed that the E. canis p43 represents the N-terminal portion of the largest immunoreactive protein described in Ehrlichia spp. with a predicted molecular mass of 153 kDa. Analysis of the recombinant N-terminal region (p43) of the p153 by protein gel electrophoresis demonstrated a larger than predicted molecular mass (∼30%), and presence of carbohydrate glycans, indicating that the p153 is a glycoprotein. A BLASTn search was performed on the E. chaffeensis genome sequence (95%), and the gene encoding the p153 ortholog was identified in E. chaffeensis. The E. canis p153 (4,263 bp) and E. chaffeensis p156 (4,389 bp) genes had similar chromosomal locations, downstream of the homologous (∼87%) deoxyguanosine-triphosphate triphosphohydrolase genes, and homologous (∼90%) intergenic sequences preceding the open reading frames. Nucleic acid sequence homology (52%) observed between the glycoprotein genes supported previous findings with regard to genetic divergence of the p43 gene fragment, and the p153 and p156 proteins had amino acid similarity of 32%. A native E. canis protein with a molecular mass of 200 kDa reacted with antisera produced against the N-terminal region (p43) of the p153, suggesting that the native protein was posttranslationally modified. Similarly, recombinant constructs of E. chaffeensis p156 migrated larger than predicted (∼200 kDa), and carbohydrate was detected on the recombinant proteins. The chromosomal location, amino acid homology, and biophysical properties support the conclusion that the p153 and p156 glycoproteins (designated gp200s) are species-specific immunoreactive orthologs.