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Ehrlichiae are obligately intracellular bacteria that reside and replicate in phagocytes by circumventing host cell defenses and modulating cellular processes, including host cell gene transcription. However, the mechanisms by which ehrlichiae influence host gene transcription have largely remained undetermined. Numerous ankyrin and tandem repeat-containing proteins associated with host-pathogen interactions have been identified in Ehrlichia species, but their roles in pathobiology are unknown. In this study, we determined by confocal immunofluorescence microscopy and by immunodetection in purified nuclear extracts that the ankyrin repeat-containing protein p200 is translocated to the nuclei of Ehrlichia- infected monocytes. Chromatin immunoprecipitation (ChIP) with DNA sequencing revealed an Ehrlichia chaffeensis p200 interaction located within host promoter and intronic Alu-Sx elements, the most abundant repetitive elements in the human genome. A specific adenine-rich (mid-A-stretch) motif within Alu-Sx elements was identified using electrophoretic mobility shift and NoShift assays. Whole-genome analysis with ChIP and DNA microarray analysis (ChIP-chip) determined that genes ( n = 456) with promoter Alu elements primarily related to transcription, apoptosis, ATPase activity, and structural proteins associated with the nucleus and membrane-bound organelles were the primary targets of p200. Several p200 target genes (encoding tumor necrosis factor alpha, Stat1, and CD48) associated with ehrlichial pathobiology were strongly upregulated during infection, as determined by quantitative PCR. This is the first study to identify a nuclear translocation of bacterially encoded protein by E. chaffeensis and to identify a specific binding motif and genes that are primary targets of a novel molecular strategy to reprogram host cell gene expression to promote survival of the pathogen.

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.

Ehrlichia canis is the primary etiologic agent of canine monocytic ehrlichiosis, a globally distributed and potentially fatal disease of dogs. We previously reported on the identification of two conserved major immunoreactive antigens, gp36 and gp19, which are the first proteins to elicit an E. canis- specific antibody response, and gp200 and p28, which elicit strong antibody responses later in the acute phase of the infection. In this report, the sensitivities and specificities of five recombinant E. canis proteins for the immunodiagnosis of E. canis infection by an enzyme-linked immunosorbent assay (ELISA) were evaluated. Recombinant polypeptides gp36, gp19, and gp200 (N and C termini) exhibited 100% sensitivity and specificity for immunodiagnosis by the recombinant glycoprotein ELISA compared with the results obtained by an indirect fluorescent-antibody assay (IFA) for the detection of antibodies in dogs that were naturally infected with E. canis . Moreover, the enhanced sensitivities of gp36 and gp19 for immunodiagnosis by the recombinant glycoprotein ELISA compared to those obtained by IFA were demonstrated with dogs experimentally infected with E. canis , in which antibodies were detected as much as 2 weeks earlier, on day 14 postinoculation. gp36 and gp19 were not cross-reactive with antibodies in sera from E. chaffeensis- infected dogs and thus provided species-specific serologic discrimination between E. canis and E. chaffeensis infections. This is the first demonstration of the improved detection capability of the recombinant protein technology compared to the capability of the “gold standard” IFA and may eliminate the remaining obstacles associated with the immunodiagnosis of E. canis infections, including species-specific identification and the lack of sensitivity associated with low antibody titers early in the acute phase of the infection.

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-than-predicted mass (∼3 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 major immunoreactive proteins of 36 and 19 kDa elicit the earliest detectable antibody responses during the acute phase of canine monocytic ehrlichiosis. Genes encoding the major immunoreactive 36-kDa protein of E. canis and the corresponding ortholog of E. chaffeensis (47 kDa) were identified and the proteins characterized. The molecular masses of the strongly immunoreactive recombinant proteins were larger than predicted (26.7 and 32.9 kDa, respectively) but were consistent with those of the corresponding native proteins (36 and 47 kDa). Similar to other reported ehrlichial immunoreactive glycoproteins, carbohydrate was detected on the recombinant expressed proteins, indicating that they were glycoproteins. Both glycoproteins (gp36 and gp47) have carboxy-terminal serine/threonine-rich tandem repeat regions containing repeats that vary in number (4 to 16 repeats) and amino acid sequence among different isolates of each species. E. canis gp36 was recognized by early acute-phase antibodies (day 14), and species-specific antibody epitopes were mapped to C-terminal nonhomologous repeat units of gp36 and gp47. Periodate treatment of recombinant gp36 reduced the antibody reactivity, and nonglycosylated synthetic peptide repeat units from E. canis gp36 and E. chaffeensis gp47 were substantially less immunoreactive than corresponding recombinant peptides, demonstrating that glycans are important epitope determinants that are structurally conserved on the recombinant proteins expressed in Escherichia coli. E. canis gp36 and E. chaffeensis gp47 were differentially expressed only on the surface of dense-cored ehrlichiae and detected in the Ehrlichia -free supernatants, indicating that these proteins are released extracellularly during infection.