Identification of a Glycosylated Ehrlichia canis 19-Kilodalton Major Immunoreactive Protein with a Species-Specific Serine-Rich Glycopeptide Epitope

Abstract

Ehrlichia canis has a small subset of major immunoreactive proteins that includes a 19-kDa protein that elicits an early Ehrlichia-specific antibody response in infected dogs. We report herein the identification and molecular characterization of this highly conserved 19-kDa major immunoreactive glycoprotein (gp19) ortholog of the Ehrlichia chaffeensis variable-length PCR target (VLPT) protein. E. canis gp19 has substantial carboxyl-terminal amino acid homology (59%) with E. chaffeensis VLPT and the same chromosomal location; however, the E. chaffeensis VLPT gene (594 bp) has tandem repeats that are not present in the E. canis gp19 gene (414 bp). Consistent with other ehrlichial glycoproteins, the gp19 protein exhibited a larger-thanpredicted mass (ϳ3 kDa), O-linked glycosylation sites were predicted in an amino-terminal serine/threonine/ glutamate (STE)-rich patch (26 amino acids), carbohydrate was detected on the recombinant gp19 protein, and the neutral sugars glucose and galactose were detected on the recombinant amino-terminal polypeptide. E. canis gp19 composition consists of five predominant amino acids, cysteine, glutamate, tyrosine, serine, and threonine, concentrated in the STE-rich patch and a carboxyl-terminal domain predominated by cysteine and tyrosine (55%). The amino-terminal STE-rich patch contained a major species-specific antibody epitope strongly recognized by serum from an E. canis-infected dog. The recombinant glycopeptide epitope was substantially more reactive with antibody than the synthetic (nonglycosylated) peptide, and periodate treatment of the recombinant glycopeptide epitope reduced its immunoreactivity, demonstrating the importance of a carbohydrate immunodeterminant(s). The gp19 protein was present on reticulate and dense-cored cells, and it was found extracellularly in the fibrillar matrix and associated with the morula membrane, the host cell cytoplasm, and the nucleus.Ehrlichia canis is a tick-transmitted obligately intracellular bacterium that causes moderate to severe and sometimes fatal disease in wild and domestic canids. The genomes of E. canis and other organisms in the genus, including Ehrlichia chaffeensis and Ehrlichia ruminantium, exhibit a high degree of genomic synteny, paralogous protein families, a large proportion of proteins with transmembrane helices and/or signal sequences, tandem repeats and ankyrin domains in proteins associated with host-pathogen interactions, and a unique serine-threonine bias associated with a potential for O glycosylation and phosphorylation (6,10,11,18). A small subset of the approximately 1,000 proteins (including hypothetical proteins) encoded by each of these genomes is recognized by antibody (8,20,25,32). Several of the major immunoreactive proteins identified and molecularly characterized are serine-rich glycoproteins that are secreted. Many of these glycoproteins have tandem repeats; however, one has numerous eukaryote-like ankyrin domains (7,20,25,27,31,35).Numerous proteins that contain tandem repeats have been identified in E. ...

A Variable-Length PCR Target Protein of Ehrlichia chaffeensis Contains Major Species-Specific Antibody Epitopes in Acidic Serine-Rich Tandem Repeats

“…This is consistent with the previously reported antibody epitope identified in E. canis gp19 (VLPT ortholog), which was also species specific (19). Furthermore, we identified similar species-specific epitopes in E. chaffeensis and E. canis protein orthologs, including gp120/gp140, gp47/gp36, and gp200s (7,16,21,38,39).…”

“…Three major epitope-containing regions were identified in the E. chaffeensis VLPT protein, in the nonidentical serine-rich repeat units R2, R3, and R4, which is consistent with the location of epitopes in other ehrlichial TR-containing proteins (7,19,21). The antibody epitope in R3, which exhibited the strongest antibody reactivity with both human sera, was localized to a 17-amino-acid N-terminal region that was highly homologous with R2 (two amino acid changes).…”

“…Hence, the identity of VLPT and the extent of the host response directed against it have remained undetermined. Recently, we described the identification and characterization of a conserved, strongly acidic major immunoreactive 19-kDa protein (gp19) in E. canis that elicits an early antibody response (19). We also concluded, based on genomic and protein analysis, that the E. chaffeensis VLPT protein was the ortholog of gp19.…”

“…Most recently, a strongly acidic 19-kDa major immunoreactive protein (gp19) of E. canis was identified. The gp19 gene has the same relative chromosomal location as and substantial amino acid homology in a C-terminal cysteine-tyrosine-rich domain to the previously reported variable-length PCR target (VLPT) protein identified in E. chaffeensis (19). The VLPT gene has 90-bp TRs that vary in number (2 to 6) in E. chaffeensis isolates; hence, it has been utilized as a molecular target for differentiation of E. chaffeensis isolates (32,35).…”

“…Furthermore, major antibody epitopes of both gp120 and gp47 have been mapped to these serine-rich acidic TRs (7,37). Similarly, the VLPT protein has three to six nonidentical serine-rich TRs (30 amino acids); however, the E. canis ortholog (gp19) lacks multiple TRs but has a serine-rich epitope-containing domain consistent in size and composition to a single VLPT repeat unit (19,32).…”