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

Abstract

Ehrlichia chaffeensis and E. canis have a small subset of tandem repeat (TR)-containing proteins that elicit strong host immune responses and are associated with host-pathogen interactions. In a previous study, we molecularly characterized a highly conserved 19-kDa major immunoreactive protein (gp19) of E. canis and identified the corresponding TR-containing ortholog variable-length PCR target (VLPT) protein in E. chaffeensis. In this study, the native 32-kDa VLPT protein was identified and the immunodeterminants defined in order to further understand the molecular basis of the host immune response to E. chaffeensis. Synthetic and/or recombinant polypeptides corresponding to various regions of VLPT were used to localize major antibody epitopes to the TR-containing region. Major antibody epitopes were identified in three nonidentical repeats (R2, R3, and R4), which reacted strongly with antibodies in sera from an E. chaffeensis-infected dog and human monocytotropic ehrlichiosis patients. VLPT-R3 and VLPT-R2 reacted most strongly with antibody, and the epitope was further localized to a nearly identical proximal 17-amino-acid region common between these repeats that was species specific. The epitope in R4 was distinct from that of R2 and R3 and was found to have conformational dependence. VLPT was detected in supernatants from infected cells, indicating that the protein was secreted. VLPT was localized on both reticulate and dense-core cells, and it was found extracellularly in the morula fibrillar matrix and associated with the morula membrane.Ehrlichia chaffeensis is a tick-transmitted, obligately intracellular bacterium which causes human monocytotropic ehrlichiosis (HME), an emerging life-threatening disease in humans, and also causes mild to severe disease in canines (28). Recently, a number of studies demonstrated that humoral immunity plays an essential role in host defenses against ehrlichial pathogens (10,33,34,36). Furthermore, a small subset of E. chaffeensis proteins, many of which contain tandem repeats (TRs), appear to be the primary targets of the humoral immune response and are considered to be the major immunoreactive proteins (4,5,15,30). However, the characteristics of the immunodeterminants that shape the humoral immune response to Ehrlichia species are not fully defined, nor has their role in protective immunity been determined.Major immunoreactive proteins of E. chaffeensis include 30). Some of these proteins (200-, 120-, 47-, and 28-kDa proteins) have been identified and molecularly characterized, including the corresponding orthologs in Ehrlichia canis respectively) (7,16,20,25,27,(38)(39)(40). 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 t...

Tyrosine-Phosphorylated Ehrlichia chaffeensis and Ehrlichia canis Tandem Repeat Orthologs Contain a Major Continuous Cross-Reactive Antibody Epitope in Lysine-Rich Repeats

“…Major antibody epitopes in the molecularly characterized TRPs have been localized to the TR regions, and these epitopes are molecularly distinct and do not elicit cross-reactive antibodies (3,13,15). This observation is consistent with the conclusion that long period tandem repeats found distributed throughout the genome evolved through independently occurring events after the divergence of the species and appear to be part of a host adaptation mechanism (5).…”

“…This finding is consistent with other major antibody epitopes that have been previously mapped to the TR regions of TRPs. Major epitopes have now been mapped to the TR regions of four TRP orthologs from E. chaffeensis and E. canis (3,13,15,19), demonstrating a prominent role for TRPs in the stimulation of antibodies against Ehrlichia spp. Antibody epitopes characterized in other TRPs are species specific; however, we found that the TRP95 and TRP75 TR regions exhibit substantial amino acid homology and the epitopes are cross-reactive.…”

“…TR epitope is cross-reactive. The TRs of previously characterized Ehrlichia TRPs are molecularly distinct and have species-specific epitopes (3,(13)(14)(15). However, the E. chaffeensis TRP75 and E. canis TRP95 have significant identity (ϳ70%) and have TRs that are identical in length (24 amino acids).…”

“…TRPs that have been identified and characterized are acidic (pI ϳ4.0), migrate abnormally during gel electrophoresis due to their acidic nature, exhibit high serine/threonine content, and contain predicted sites for phosphorylation, and some are differentially expressed on dense-cored ehrlichiae (3,15,24). Major antibody epitopes in the molecularly characterized TRPs have been localized to the TR regions, and these epitopes are molecularly distinct and do not elicit cross-reactive antibodies (3,13,15).…”

“…Three pairs of tandem repeat protein (TRP) orthologs have been molecularly characterized in E. chaffeensis and E. canis, including TRP120/ TRP140, TRP47/TRP36, and TRP32/TRP19 and major continuous species-specific epitopes mapped to serine-rich acidic tandem repeat (TR) regions (3,13,15). Other major immunoreactive proteins that have been molecularly characterized include an ankyrin repeat protein (Ank200) and an outer membrane protein family (OMP-1) (14,22,23,25).…”