Microbial Immunity and Vaccines

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

Jere W. McBride, Xiaofeng Zhang, Abdul Wakeel, Jeeba A. Kuriakose
A. Camilli, Editor
DOI: 10.1128/IAI.01347-10

ABSTRACT

A small subset of major immunoreactive proteins have been identified in Ehrlichia chaffeensis and Ehrlichia canis, including three molecularly and immunologically characterized pairs of immunoreactive tandem repeat protein (TRP) orthologs with major continuous species-specific epitopes within acidic tandem repeats (TR) that stimulate strong antibody responses during infection. In this study, we identified a fourth major immunoreactive TR-containing ortholog pair and defined a major cross-reactive epitope in homologous nonidentical 24-amino-acid lysine-rich TRs. Antibodies from patients and dogs with ehrlichiosis reacted strongly with recombinant TR regions, and epitopes were mapped to the N-terminal TR region (18 amino acids) in E. chaffeensis and the complete TR (24 amino acids) in E. canis. Two less-dominant epitopes were mapped to adjacent glutamate/aspartate-rich and aspartate/tyrosine-rich regions in the acidic C terminus of E. canis TRP95 but not in E. chaffeensis TRP75. Major immunoreactive proteins in E. chaffeensis (75-kDa) and E. canis (95-kD) whole-cell lysates and supernatants were identified with TR-specific antibodies. Consistent with other ehrlichial TRPs, the TRPs identified in ehrlichial whole-cell lysates and the recombinant proteins migrated abnormally slow electrophoretically a characteristic that was demonstrated with the positively charged TR and negatively charged C-terminal domains. E. chaffeensis TRP75 and E. canis TRP95 were immunoprecipitated with anti-pTyr antibody, demonstrating that they are tyrosine phosphorylated during infection of the host cell.

INTRODUCTION

Substantial progress has been made in defining the molecular immunodeterminants of Ehrlichia species, and many of the pathogen proteins targeted by the host immune response are secreted tandem repeat-containing proteins. 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). Recently, putative lipoproteins have been identified in the E. chaffeensis genome, and dogs immunized with four of these proteins develop specific antibody and cell-mediated immune responses (7).

TRPs that have been identified and characterized are acidic (pI ∼4.0), 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). 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). Although the function of TRPs during infection is not completely understood, they are secreted and appear to be effector proteins involved in complex molecular interactions with host proteins associated with numerous cellular processes (21, 29). E. chaffeensis TRP47 and TRP120 are differentially expressed on dense-cored ehrlichiae and the TRP47 interacts with a variety of host cell proteins, including polycomb group ring finger protein (PCGF5) and immunoglobulin light chains (27).

The kinetics of the host immune response indicates that TRPs are highly expressed early in infection and accessible to the host antibody response (17, 18). In fact, microarray analysis of E. chaffeensis gene expression in monocytes revealed that TRP47 had the highest expression levels of any ehrlichial gene (11). In dogs experimentally infected with E. canis, TRP19 and TRP36 (TRP47 ortholog) are among the first ehrlichial proteins to elicit antibodies (2, 17). Moreover, antibodies directed at the major TR antibody epitope of the E. chaffeensis TRP120 as well as the hypervariable region of the OMP-1g outer membrane protein have been shown to reduce the bacterial burden in E. chaffeensis-infected mice, demonstrating that they are targets of protective immunity and have important functional roles that can be blocked by antibody (10, 12), possibly including, but not limited to inhibiting porin activity or molecular interactions facilitating binding and internalization (8, 9, 24).

There are several major immunoreactive proteins that have not been identified and molecularly characterized in E. chaffeensis and E. canis. In the present study, we report the identification and molecular characterization of a fourth pair of TRP orthologs of E. chaffeensis (TRP75) and E. canis (TRP95) that are strongly immunoreactive and contain a major cross-reactive continuous epitope in the TR region. We also identified a second major epitope in the acidic C-terminal domain of the E. canis TRP95 that was not found in the E. chaffeensis TRP75. Similar to other characterized TRPs, the TRP75 and TRP95 migrate abnormally during gel electrophoresis but differ in that they have a basic (pI 10) lysine-rich TR region. The E. chaffeensis TRP75 has been previously identified as a putative lipoprotein (7), but the molecular characteristics, epitopes, and identity of the native protein and E. canis ortholog investigated in the present study were not previously determined.

MATERIALS AND METHODS

Ehrlichia propagation and purification.E. chaffeensis (Arkansas strain) and E. canis (Jake strain) were propagated and purified as previously described (31). The fractions containing bacteria were frozen and utilized as antigen and DNA sources.

PCR amplification of the Ehrlichia TRP genes.Genes encoding TRPs, including Ecaj_0472 (TRP95), were identified in the genome sequence of E. canis and previously reported (16). Oligonucleotide primers for the amplification of the E. chaffeensis TRP75 and E. canis TRP95 gene fragments were designed manually, or by PrimerSelect (Lasergene v8.1; DNAStar, Madison, WI) according to the sequences in GenBank (accession numbers U49426 and NC_007354, respectively) and synthesized (Sigma-Genosys, Woodlands, TX) (Table 1). Gene fragments corresponding to the N- and C-terminal and TR regions and complete open reading frames were amplified by PCR (Fig. 1A and 2A). The E. chaffeensis TRP75-TR contained 10.5 tandem repeats, and E. canis TRP95-TR contained 12.5 tandem repeats (Fig. 1A and 2A).

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Table 1.

Oligonucleotide primers used for amplification of E. chaffeensis and E. canis TRPs

Fig. 1.
Fig. 1.

Schematic representation of E. chaffeensis TRP75, recombinant proteins, and peptides and immunoreactivity determined by Western immunoblotting and ELISA. (A) E. chaffeensis TRP75 domains, locations of TRs (numbers of amino acids and pI values are given in parentheses; R = repeat), recombinant proteins and synthetic peptides used for epitope mapping. Variable amino acids in each TR are shown in boldface and underlined. (B) Identification of native E. chaffeensis TRP75 by Western immunoblotting (left). E. chaffeensis whole-cell lysate derived from E. chaffeensis-infected DH82 cells reacted with anti-E. chaffeensis canine sera (lane 1) and rabbit anti-TRP75 (lane 2) antibody and DH82 lysate (Ctrl). Immunoreactivity of recombinant E. chaffeensis TRP75 protein fragments by Western immunoblotting (right). SDS-PAGE and total protein staining of purified recombinant TRP75 fragments (N terminus [N1, N2], tandem repeat region [TR], and C terminus [C1]) (center) and the corresponding Western immunoblot probed with HME patient sera (right). Human or canine sera did not recognize thioredoxin, and the normal human and canine sera did not react with these recombinant proteins by Western immunoblotting (data not shown). M, molecular masses (kilodaltons). (C) Immunoreactivity by ELISA of recombinant TRP75 fragments with three HME patient and anti-E. chaffeensis canine sera. Immunoblot and ELISA bound antibodies were detected with alkaline phosphatase-labeled anti-human or dog IgG(H+L).

Fig. 2.
Fig. 2.

Schematic representation of E. canis TRP95, recombinant proteins, and peptides and immunoreactivity determined by Western immunoblotting and ELISA. (A) E. canis TRP95 domains, locations of TRs (the number of amino acids and pI values are given in parentheses; R = repeat), recombinant proteins, and synthetic peptides used for epitope mapping. Variable amino acids in each TR are shown in boldface and underlined. (B) Identification of native E. canis TRP95 by Western immunoblotting (left). E. canis whole-cell lysate derived from E. canis-infected DH82 cells reacted with anti-E. canis canine sera (lane 1) and rabbit anti-TRP95 (lane 2) antibody. Antibody did not react with DH82 lysate (Ctrl). Immunoreactivity of recombinant proteins of E. canis TRP95 by Western immunoblotting (right). SDS-PAGE and total protein staining of purified recombinant TRP95 fragments (N terminus [N], tandem repeat region [TR], C terminus [C, C1, and C2]) (center) and the corresponding Western immunoblot probed with anti-E. canis canine sera (right). Canine sera did not recognize thioredoxin, and normal canine sera did not react with these recombinant proteins by Western immunoblotting (data not shown). M, molecular masses (kilodaltons). (C) Immunoreactivity by ELISA of recombinant TRP95 fragments with three anti-E. canis canine sera. Immunoblot and ELISA bound antibodies were detected with alkaline phosphatase-labeled anti-human or dog IgG(H+L).

Expression and purification of recombinant proteins.The amplified PCR products were cloned directly into the pBAD/Thio-TOPO expression vector (Invitrogen, Carlsbad, CA) and transformed E. coli TOP10 cells (Invitrogen). The resulting transformants were screened by PCR for correctly oriented inserts, and plasmids from the positive transformants were isolated and sequenced to verify the inserts at the University of Texas Medical Branch Protein Chemistry Core Laboratory. Recombinant protein expression was performed for 3 h after induction with 0.2% arabinose, and proteins were purified under native conditions using HisSelect columns (Sigma, St. Louis, MO).

Synthetic peptides.For the E. chaffeensis TRP75, five overlapping peptides corresponding to three repeat units (R1, R2, and R5) and four overlapping peptides corresponding to a single C-terminal domain (C2) were commercially synthesized (Bio-Synthesis, Lewisville, TX) (Fig. 1A). For E. canis TRP95, nine overlapping peptides corresponding to three different repeat regions (R1, R2, and R3) and seven overlapping peptides corresponding to a single C-terminal unit (C2) were synthesized (Bio-Synthesis) (Fig. 2A). All peptides were supplied as a lyophilized powder and resuspended in molecular-biology-grade water (1 mg/ml).

Antisera.Anti-E. canis canine sera were obtained from two experimentally infected dogs and one from a naturally infected dog described previously (14). Human monocytotropic ehrlichiosis (HME) patient sera were kindly provided by Focus Technologies (Cypress, CA) and William Nicholson at the Centers for Disease Control and Prevention (Atlanta, GA). Rabbit anti-E. chaffeensis TRP75 and anti-E. canis TRP95 antibodies were generated against the synthetic KLH-conjugated peptides located in the epitope-containing region of each respective repeat unit (E. chaffeensis TRP75, DVKDNKPSDVKLPVIKAE; E. canis TRP95-R2, DDSKLPVIKVEDKSKLQDTKDKKR) by a commercial vendor (Biosynthesis).

Gel electrophoresis and Western immunoblotting.Purified E. chaffeensis or E. canis whole-cell lysates or recombinant proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose, and Western immunoblotting was performed as previously described (17), except that primary antisera were diluted as follows: dog, 1:100; human, 1:200; and rabbit, 1:1000.

ELISA.Enzyme-linked immunosorbent assay (ELISA) plates (PolySorp or MaxiSorp; Nunc, Roskilde, Denmark) were coated with recombinant proteins or synthetic peptides (0.5 μg/well; 50 μl) suspended in phosphate-buffered saline (PBS; pH 7.4) as previously described (13). Briefly, proteins and peptides were absorbed for 1 h at room temperature with gentle agitation and subsequently washed thrice with 200 μl of Tris-buffered saline containing 0.2% Tween 20 (TBST). The plates were blocked with 100 μl of 10% equine serum (Sigma) in TBST for 1 h at room temperature with agitation and then washed. Convalescent dog or human sera diluted (1:100 or 1:200, respectively) in 10% equine serum-TBST were added to each well (50 μl), followed by incubation at room temperature for 1 h with gentle agitation. The plates were washed four times, and 50 μl of alkaline phosphatase-labeled goat anti-canine or human IgG(H+L) secondary antibody (Kirkegaard & Perry Laboratories, Gaithersburg, MD) diluted 1:5,000 in 10% equine serum-TBST was added, followed by incubation for 1 h at room temperature. The plates were washed four times, and 100 μl of substrate (BluePhos; Kirkegaard & Perry Laboratories) was added to each well. The plates were incubated in the dark for 30 min with agitation; the optical density (OD) was determined on a microplate reader (VersaMax; Molecular Devices, Sunnyvale, CA) at A650, and the data were analyzed using SoftmaxPro v4.0 (Molecular Devices). OD readings represent the mean OD for three wells (±standard deviations) after subtracting the OD value of normal serum (dog or human).

Mass spectrometry.Mass spectrometry was performed using a matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometer (Voyager-DE STR; Applied Biosystems) at the University of Texas Medical Branch Mass Spectrometry Core Laboratory as previously described (28).

Detection and immunoprecipitation of pTyr TRPs.Whole-cell lysates were prepared as described previously (28) with some modifications. Briefly, 107 normal cells and E. chaffeensis- or E. canis-infected cells (at 3 days postinfection) were collected (500 × g, 5 min), washed twice in ice-cold PBS, resuspended in 1 ml of ice-cold radioimmunoprecipitation assay lysis buffer (Pierce, Rockford, IL) that contained complete Mini protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN), phosphatase inhibitor cocktail (Pierce), 5 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride-sodium fluoride-sodium orthovanadate, and incubated for 20 min on ice. Cell lysates were prepared by sonication of cells for 1 min on ice. Lysates were collected by centrifugation at 12,000 × g for 10 min at 4°C. Tyrosine phosphorylation on the native TRPs was detected by Western immunoblot using anti-phosphotyrosine antibody (PY99; Santa Cruz Biotechnology, Santa Cruz, CA) as previously described (28). Immunoprecipitations were performed as previously described (28) with the following modifications. Whole-cell lysates were precleared with 50 μl of protein A/G Ultralink resin 50% slurry (Pierce) and 20 μl of mouse IgG2b monoclonal antibody (R&D Systems, Minneapolis, MN) for 1 h at 4°C. The lysates were centrifuged briefly, and the supernatant was collected. The supernatants containing 500 μg of protein (1 mg/ml) were incubated with 2 μg (for E. chaffeensis) or 10 μg (for E. canis) of either mouse anti-pTyr monoclonal antibody or mouse IgG2b monoclonal antibody (isotype control) with gentle mixing for 16 h at 4°C. The immunocomplexes were precipitated with 30 μl of protein A/G resins (50% slurry) by gentle mixing for 2 h at 4°C. The resin beads were centrifuged briefly at 1,000 × g for 30 s and then washed four times with lysis buffer and once with PBS before being boiled for 5 min in 30 μl of 2× LDS sample buffer with 1× sample reducing agent (Invitrogen).

Confocal immunofluorescence microscopy.Expression of the TRPs on E. chaffeensis- and E. canis-infected cells was determined using confocal immunofluorescence microscopy as previously described (27).

Sequence analysis.Amino acid sequence alignments of E. chaffeensis TRP75 and E. canis TRP95 were performed with MegAlign (Lasergene v5.08; DNAStar). The major epitopes of TRP75 and TRP95 were examined for homology to other proteins by using the protein-protein basic local alignment search tool (BLAST [http://www.ncbi.nlm.nih.gov/BLAST]). The predicted tyrosine phosphorylation sites were determined using NetPhos 2.0, and secretion prediction was determined by SecretomeP 2.0 at the Center for Biological Sequence Analysis (http://www.cbs.dtu.dk/index.shtml).

RESULTS

E. chaffeensis and E. canis TRP characteristics.Overall, E. chaffeensis TRP75 and E. canis TRP95 are slightly acidic proteins (pIs of 5.5 and 6.4, respectively). The three different domains of these proteins were identified based on location and pI (C- and N-terminal regions and TR). There were substantial differences in the pI among the N- and C-terminal regions and TR regions. The N-terminal region of both TRPs was more neutral (pI 6.1 and 8.4, respectively), the C-terminal regions were acidic (pI 4.0 and 4.2), and the TR regions were basic (pI 10 and 9.9) (Fig. 1A and 2A). Lysine (17%), aspartate (14%) and valine (11%) were the three most frequently occurring amino acids in the E. chaffeensis TRP75 (Table 2). E. canis TRP95 had slightly higher frequencies of lysine (19%) and aspartate (16%) but a lower frequency of valine (8%) (Table 2). These three amino acids accounted for 42% of the entire amino acid content of the TRP orthologs. The frequency of lysine was substantially higher in the TR region with a composition of 34% in the E. chaffeensis TR and 27% in E. canis, thus increasing the frequency of these three amino acids to 65% of the total amino acids in the TR of E. chaffeensis and 56% in the E. canis TR. The high frequency of lysine in the TR regions was associated with the basic pI (∼10) of the TR region. Notably, a high frequency of tyrosine and aspartate residues was identified in a 24-amino-acid conserved domain found in both TRP orthologs at the C-terminal end of the protein (Fig. 3B and D). BLAST analysis determined that this domain had homology to multiple serine/threonine protein kinases (e.g., PrkC of Clostridium spp. and PknB of Corynebacterium).

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Table 2.

Predicted and observed molecular masses and amino acid analysis of E. chaffeensis and E. canis TRP

Fig. 3.
Fig. 3.

Immunoreactivity of overlapping synthetic peptides spanning the E. chaffeensis TRP75 and E. canis TRP95 repeat units and C termini by ELISA. (A and B) E. chaffeensis TRP75 peptides reacted with sera from three HME patients and anti-E. chaffeensis canine sera. Positive control (Ctrl), E. chaffeensis TRP75-R5 peptide (Patient Ctrl), and E. canis TRP95-C2 peptide (Dog Ctrl) reacted with HME patient and canine sera, respectively. (C and D) E. canis TRP95 peptides reacted with sera from three E. canis-infected dogs. The OD readings represent the means of three wells (± standard deviation) with the OD of nonimmune serum subtracted. Variable amino acids in the TR are underlined (A and C), and conserved 24-amino-acid aspartate-tyrosine-rich C-terminal domains are underlined in panels B and D.

Six variations in the amino acid sequence of the E. chaffeensis TRs were identified in the 10.5 repeat TR region. R1 was the most frequent (n = 4) repeat unit, followed by R2 (n = 2) and R3 (n = 2) (Fig. 1A). Differences in these repeats were specific to substitutions in three hypervariable amino acids (3, 6, and 21) of each repeat (Fig. 1A). In E. canis, five different repeat variations were identified in the 12.5 repeat TR region. R2 was found most frequently (n = 8), followed by R3 (n = 2) (Fig. 2A). Like E. chaffeensis, differences in these repeats were specific to substitutions in two variable amino acids (7 and 8) (Fig. 2A). No significant homology was found between the TRs and other known ehrlichial proteins or proteins from other organisms.

Identification of native TRPs.TRPs in ehrlichial whole-cell lysates and supernatant from infected cells were detected by Western immunoblot using peptide-specific antisera against the respective repeat units. For E. chaffeensis, predominant native proteins of ca. 75 and 60 kDa were identified, and several smaller protein bands (ca. 45 to 55 kDa) also reacted (Fig. 1B). The proteins that reacted with anti-E. chaffeensis TRP antibody corresponded to bands detected with polyclonal E. chaffeensis antisera from an infected dog (Fig. 1B). E. canis anti-TRP antibody reacted with an ∼95-kDa protein and several other lower molecular mass proteins (ca. 60 to 70 kDa) (Fig. 2B). Proteins in whole-cell lysates that reacted with E. canis TRP-specific antibody were also recognized by polyclonal antibody from an E. canis-infected dog (Fig. 2B). Preimmune serum did not react with Ehrlichia proteins (data not shown).

Recombinant TRPs and fragment immunoreactivity.Recombinant proteins representing the different TRP domains were cloned, expressed, purified, and reacted by Western immunoblotting and ELISA to determine the immunoreactivity. The C2 fragment of E. chaffeensis could not be expressed in E. coli. Of the expressed recombinant fragments, TR and C-terminal E. chaffeensis and E. canis protein fragments migrated larger than predicted by SDS-gel electrophoresis (Fig. 1B and 2B; summarized in Table 2); however, N-terminal fragments migrated at their predicted masses. As determined by Western immunoblotting and ELISA, the TR region of both TRPs exhibited strong reactivity with antibody (Fig. 1B and 2B). The E. canis C-terminal fragment (C2) also reacted strongly with antibody by Western immunoblotting and ELISA (Fig. 2B and C). Conformational epitopes were not identified by ELISA in fragments that were not reactive by Western immunoblotting. Thus, major continuous antibody epitopes were localized in the TR regions of both TRPs, and a second continuous epitope was localized in the C-terminal region of E. canis TRP95.

Mass spectrometry of recombinant TRPs.We observed that the native and recombinant TRPs migrate larger than predicted by SDS-gel electrophoresis. We have previously determined that the larger than predicted masses associated with the acidic TR regions of ehrlichial TRPs are associated with the acidic nature of the proteins and the lack of SDS binding (28). We performed MALDI-TOF on the E. chaffeensis and E. canis TRP recombinant fragments that exhibited abnormal electrophoretic migration. The largest abnormal mass was associated with the basic TR region of both proteins, which was ∼10 kDa larger than predicted (Fig. 1B and 2B; summarized in Table 2). However, according to mass spectrometry the masses of the TR regions of both TRPs were consistent with the predicted masses demonstrating that these proteins were not posttranslationally modified (Table 2). Similarly, recombinant fragments that represented the acidic C-terminal region (see Fig. 1A and 2A) also migrated as ca. 4- to 9-kDa larger masses than predicted, but mass spectrometry demonstrated that the masses of these proteins were consistent with the predicted mass (Table 2).

Peptide epitope mapping.By Western immunoblotting and ELISA the TR regions of both TRPs were strongly reactive. We used peptide mapping to further characterize the epitope(s) contained within these regions. The immunoreactivity of three E. chaffeensis TRP75 repeat units (R1, R2, and R5; 24 amino acids; see Fig. 1A) was examined by using overlapping peptides (Fig. 3A). Three HME patient sera reacted strongly and similarly with R1 (most frequent TR unit), R5 and the smaller R1N and R5N (18 amino acids) peptides (Fig. 1A and 3). R2 and related peptides (R2N and R2C) were inconsistently and more weakly recognized by patient sera, suggesting that two amino acid substitutions PS to ST (amino acids 13 and 14) in the TR R2 are important epitope determinants (Fig. 1A and 3A). Sera from a dog experimentally infected with E. chaffeensis also reacted with the TRP75 TR peptides (Fig. 3A). An epitope was not detected in the E. chaffeensis C1 protein (Fig. 3B and see Fig. 1). We were unable to express the E. chaffeensis C2 as a recombinant protein; therefore, peptides (C2-1 to C2-4) were used to probe the 82-amino-acid domain for epitopes (see Fig. 1A). E. chaffeensis patient and dog sera did not react with any peptides from this domain (Fig. 3B).

The E. canis TRP95 TR region consists of 12.5 TRs, with the predominant type being R2 in addition to three other variations that differ primarily in a 2-amino-acid sequence variation (Fig. 2A). Peptides representing the three TRs (24 amino acids) were reacted with canine sera from E. canis-infected dogs (Fig. 3). All TR peptides were reactive, but R1 and R3 were recognized similarly and more strongly by all dogs (Fig. 3C). Overlapping peptides (18-mer) from the N-terminal and C-terminal regions of the three most frequent repeat types (R1, R2, and R3) were examined, and the C-terminal 18 amino acids were more strongly recognized by antibodies in canine sera (2 of 3) (Fig. 3C). The N-terminal peptides also reacted strongly with antibodies in canine sera in two of three dogs (Fig. 3C). An epitope was localized to the C-terminal region of the E. canis TRP95 (see Fig. 2B), and three large (29 to 33 amino acids) overlapping peptides were used to further map the epitope (Fig. 3D). Antibodies from canine sera reacted with all three larger peptides, but peptides C2-2 and C2-3 were recognized most strongly by all canine sera. Four smaller peptides (18 amino acids) within the region represented by C2-2 and C2-3 were used to map this epitope. Only peptide C2-C3 reacted strongly with all canine sera (Fig. 3D).

TR epitope is cross-reactive.The TRs of previously characterized Ehrlichia TRPs are molecularly distinct and have species-specific epitopes (3, 13–15). However, the E. chaffeensis TRP75 and E. canis TRP95 have significant identity (∼70%) and have TRs that are identical in length (24 amino acids). Antibodies produced against the E. canis TR peptide (R2) were reacted with recombinant E. chaffeensis and E. canis TR regions and whole-cell lysates (Fig. 4A). In E. canis whole-cell lysates, a protein (∼95 kDa) reacted strongly with the TR (R2) antibody and multiple smaller proteins (ca. 60 to 70 kDa) were also reactive (Fig. 4A). The anti-E. canis TR (R2) antibody also reacted with proteins in E. chaffeensis whole-cell lysates that were consistent with proteins identified with E. chaffeensis TR-specific antibody (Fig. 4A; see Fig. 1B). E. chaffeensis TR peptides (R1, R2, and R5) exhibited similar cross-reactivity with three E. canis-infected dog sera (Fig. 4B), and E. canis TR peptides (R1, R2, and R3) were recognized by antibodies in three HME patient sera (Fig. 4C). As observed with homologous E. canis sera (Fig. 3A), the E. canis R2 peptide was not as strongly recognized by one HME patient.

Fig. 4.
Fig. 4.

(A) Cross-reactive tandem repeat epitope of E. chaffeensis TRP75 and E. canis TRP95 demonstrated by Western immunoblotting and ELISA. E. canis (E.ca)- and E. chaffeensis (E.ch)-infected DH82 cell lysates and recombinant E. canis TRP95 (E.caTR) and E. chaffeensis TRP75 (E.chTR) tandem repeat regions reacted with rabbit anti-E. canis TRP95 peptide (R2) serum. Anti-TRP95 antibody did not react with the fusion protein thioredoxin (Thio) or uninfected DH82 lysate (DH82). Molecular mass makers are shown on the left in kilodaltons. (B and C) Cross-reactivity of E. chaffeensis TR peptides R1, R2, and R5 with sera from dogs experimentally infected with E. canis (B) and E. canis TR peptides R1, R2, and R3 with HME patient sera (C) as determined by ELISA.

Tyrosine phosphorylation of the E. chaffeensis TRP75 and E. canis TRP95.The E. chaffeensis and E. canis TRP have tyrosine-rich C-terminal domains, suggesting that the proteins may be tyrosine phosphorylated during infection. We probed E. chaffeensis and E. canis whole-cell lysates with anti-E. chaffeensis TRP75- and E. canis TRP95-specific antibodies and anti-pTyr antibodies. Two proteins with similar molecular masses to those identified with the TRP75 antibody also reacted with anti-pTyr antibody (Fig. 5A). The E. canis TRP95 could not be identified by pTyr Western immunoblotting because comigrating host cell proteins reacted with the antibody (data not shown). Predicted tyrosine phosphorylation sites (n = 5) were identified by NetPhos 2.0 in the N- and C-terminal regions of the TRPs. To confirm the tyrosine phosphorylation of the E. chaffeensis TRP75 and E. canis TRP95, we immunoprecipitated proteins from E. chaffeensis- and E. canis-infected cells (THP-1 and DH82 cells, respectively) with anti-pTyr and detected the immunoprecipitated proteins with TRP75- and TRP95-specific antibodies. Coimmunoprecipitation of TRP75 and TRP95 with anti-pTyr demonstrated enrichment of TRP75 and TRP95 compared to that of isotype control (mouse monoclonal IgG2b) antibodies (Fig. 5B).

Fig. 5.
Fig. 5.

Tyrosine phosphorylation of E. chaffeensis TRP75 and E. canis TRP95. (A) Western blot of one-dimensional SDS-PAGE of whole-cell lysates from normal (N) and E. chaffeensis-infected THP-1 (Ech) lysates probed with anti-pTyr antibody or anti-TRP75. Molecular mass markers are indicated on the left (in kilodaltons). (B) Immunoprecipitation of TRP75 and TRP95 with anti-pTyr antibody. Whole-cell lysates from E. chaffeensis-infected THP-1 cells (Ech) and E. canis-infected DH82 cells (Eca) were immunoprecipitated with an anti-pTyr mouse monoclonal antibody (IP-pTyr) and isotype control mouse monoclonal IgG2B antibody (IP-control). Immunoprecipitates were analyzed by SDS-PAGE followed by transfer of proteins and Western immunoblotting with an anti-TRP75 and anti-TRP95 specific antibody, respectively.

Confocal immunofluorescence microscopy.Expression of two well-characterized E. chaffeensis TRPs (120 and 47) is observed only on dense-cored ehrlichiae (3, 24). We examined E. chaffeensis TRP75 expression on E. chaffeensis cultivated in THP-1 cells and observed that TRP75 was not differentially expressed and was found on all ehrlichiae that reacted with a pan Ehrlichia antibody (Dsb) (20) (Fig. 6).

Fig. 6.
Fig. 6.

Immunofluorescent confocal microscopy of E. chaffeensis-infected THP-1 cells fixed and stained with DAPI (blue), anti-TRP75 (green), and anti-Dsb (red) antibodies demonstrated colocalization of TRP75 and Dsb. The double immunofluorescence labeling of E. chaffeensis-infected THP-1 cells revealed that all of the ehrlichiae are dually labeled with TRP75 and Dsb (expressed on DC and RC).

DISCUSSION

Most of the major immunoreactive proteins of E. canis described in 2003 by McBride et al. and corresponding orthologs in E. chaffeensis have been molecularly and immunologically characterized (3, 13–15, 19, 22). It is well established that the host immune response against Ehrlichia spp. is directed at a small subset of proteins, the majority of which are known to contain tandem repeats. The molecularly characterized TRPs have single major species-specific continuous antibody epitopes and are secreted. In addition to being primary targets of the host antibody response, molecular interactions between the TRP47 and the host cell have been described, and other TRPs such as the TRP120 appear to play important roles as effector proteins (27, 29). In the present study, we identified and molecularly characterized another pair of TRP orthologs in E. chaffeensis and E. canis that were previously identified as putative lipoproteins (7).

Interestingly, these TRPs have two significant distinct differences from other TRPs that have been characterized. These differences include a TR region that is highly basic rather than acidic, and the major antibody epitopes (mapped to the TR regions) are cross-reactive. Overall, the E. chaffeensis TRP75 and E. canis TRP95 are slightly acidic (pI 5.5 to 6.5), but both contain basic (pI 10) TR regions attributed to high lysine content. The highly basic TR region is flanked by a neutral N-terminal region (∼140 amino acids) and acidic C-terminal region (170 amino acids; pI ∼4.0). Consistent with other TRPs, the native TRP orthologs in this report migrated electrophoretically with larger than the predicted masses. Furthermore, the recombinant TR and C-terminal regions also migrated electrophoretically with larger than predicted masses, We have previously reported the relationship between acidic TRPs and abnormal migration (28) and demonstrated that these charged regions are associated with larger electrophoretic masses, a characteristic associated with incomplete SDS binding (6, 28). We observed abnormal migration of recombinant protein fragments representing the TR region (pI 10) and the acidic (pI 4) C-terminal region, indicating that each of these regions contributes to the abnormal electrophoretic mobility. However, mass spectrometry confirmed that the recombinant protein fragments had masses that were consistent with their predicted masses, indicating an absence of posttranslational modification. The abnormal migration of the acidic region is best explained by incomplete binding of SDS. However, abnormal migration of the positively charged lysine-rich tandem repeat region is likely due to an alternative explanation put forward in a previous study that suggests differences in the shape of proteins (disorder) or SDS-protein complexes may also arise from their intrinsic differences in charge (1, 30). Another basic repetitive sequence protein, pig heart calpastatin, migrates abnormally slow by SDS-PAGE, an observation attributed to charged amino acids (26).

A previous study identified a lipobox sequence (LVLIFCFVISCSNK) within the N-terminal region of the E. chaffeensis TRP75 (7), and we identified a similar sequence in the E. canis TRP95 (LILIFSFVVSCSNK), suggesting that these TRPs may be modified with lipid. However, lipid has not been conclusively detected on these TRPs (7). In addition to this potential modification, we determined that the native E. chaffeensis TRP is tyrosine phosphorylated, presumably by host tyrosine kinases. The 75- and 60-kDa proteins identified by anti-E. chaffeensis TRP75 antiserum were both strongly reactive with anti-phosphotyrosine antibody. A tyrosine-rich C-terminal domain conserved between TRP75 and TRP95 had three predicted tyrosine phosphorylation sites, suggesting that this region is modified by phosphate. This finding is consistent with our previous reports demonstrating that TRP47 is also tyrosine phosphorylated (28). The TRP47 interacts with the host cell tyrosine kinase Fyn, and it appears that TRP75 may also be secreted to a location where it interacts with host cell tyrosine kinase(s). These observations suggest that TRP75 and TRP95 interact with host cell kinases and potentially signaling pathways, but further studies are needed to better understand the significance of this finding as it relates to ehrlichial pathobiology.

Major continuous cross-reactive antibody epitopes were identified in the TR regions of TRP95 and TRP75. 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. Although the tandem repeats appeared after the divergence of the two species (4), the TR regions in TRP95 and TRP75 are more conserved than those of other TRPs, suggesting these orthologs coevolved and thus may be associated with an important conserved function. Cross-reactive antibodies between Ehrlichia spp. are in part explained by immune responses directed at these homologous epitopes. Whether the TRP75 and TRP95 epitopes elicit cross-protective antibodies remains to be determined.

The TR-specific antibodies produced against the E. chaffeensis and E. canis TRPs identified in the present study react with multiple protein bands in native whole-cell lysates by Western immunoblots that are also reactive with antibodies in polyclonal anti-Ehrlichia serum. Thus, it appears that these TRPs are proteolytically processed during infection and are represented by the smaller immunoreactive proteins. The largest protein bands identified by immunoblotting appear to represent full-length protein, and major secondary bands (55 kDa for TRP75 and 65 kDa for TRP95) are consistent with the masses of these proteins without the C-terminal regions (see observed masses Table 1). Furthermore, all of these proteins react with anti-TR antibody, indicating that the TR region is present in these processed fragments. The reasons for protein processing are not clear and need to be investigated further in order to understand the significance.

During ehrlichial infection, the organism has two defined morphological forms, the dense-cored cell (DC) and reticulate cell (RC), that have been well characterized. The DC is the infectious form of E. chaffeensis and can be distinguished phenotypically by the expression of two TRPs: TRP120 and TRP47. In contrast, TRP32 is present on both forms of ehrlichiae. In the present study we determined that TRP75 is expressed on both DCs and RCs of E. chaffeensis, which is consistent with the expression of TRP32. TRP120, TRP47, and TRP32 are secreted, and the TRP47 and TRP120 have defined molecular pathogen-host interactions (27). We did not determine that TRP75 or TRP95 are secreted, but the fact that the E. chaffeensis TRP75 is phosphorylated and predicted to be secreted by a leaderless secretion mechanism suggests that it is secreted and functions as an effector protein.

In the present study, we identified a new pair of TRP orthologs, adding to an expanding group of TRPs that are strongly recognized by the host immune response; moreover, some of these TRPs are involved in novel host-pathogen interactions. Although the functions of TRP75 and TRP95 are not well understood, it is likely that these proteins play a critical role in ehrlichial survival in the phagocyte. Further investigations are needed to fully determine the role of each TRP in ehrlichial adaptation and survival in both mammalian and arthropod hosts.

ACKNOWLEDGMENTS

This study was supported by grant R01 AI 071145 from the National Institute of Allergy and Infectious Diseases (NIAID) and by the Clayton Foundation for Research. J.A.K. was supported by NIAID Biodefense training grant T32 AI060549.

We thank Simone Miyashiro for technical assistance and David Walker and Xue-Jie Yu for reviewing the manuscript and providing helpful suggestions.

FOOTNOTES

    • Received 21 December 2010.
    • Returned for modification 22 January 2011.
    • Accepted 10 May 2011.
    • Accepted manuscript posted online 23 May 2011.

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