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Infection and Immunity, April 2008, p. 1572-1580, Vol. 76, No. 4
0019-9567/08/$08.00+0     doi:10.1128/IAI.01466-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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

Tian Luo,1 Xiaofeng Zhang,1 Abdul Wakeel,1 Vsevolod L. Popov,1 and Jere W. McBride1,2,3,4*

Departments of Pathology,1 Microbiology and Immunology,2 Center for Biodefense and Emerging Infectious Diseases,3 Sealy Center for Vaccine Development, University of Texas Medical Branch, Galveston, Texas 775554

Received 1 November 2007/ Returned for modification 5 December 2007/ Accepted 10 January 2008


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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.


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INTRODUCTION
 
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 200-, 120-, 88-, 55-, 47-, 40-, 28-, and 23-kDa proteins (5, 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 (200-, 140-, 36-, and 28-kDa proteins, respectively) (7, 16, 20, 25, 27, 38-40). 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 to 6) in E. chaffeensis isolates; hence, it has been utilized as a molecular target for differentiation of E. chaffeensis isolates (32, 35). Although E. canis gp19 is strongly immunoreactive, the full extent of immunoreactivity and the molecular mass of native E. chaffeensis VLPT are unknown.

Many of the major immunoreactive proteins of E. chaffeensis and E. canis are serine-rich TR-containing proteins, including two pairs of orthologs (gp120/gp140 and gp47/gp36) (7, 16, 21, 38, 39). E. chaffeensis gp120 and gp47 are major immunoreactive proteins that are expressed differentially on the surfaces of dense-core ehrlichiae and are secreted (7, 29). The gp120 protein contains two to five nearly identical serine-rich TRs with 80 amino acids each, and gp47 has carboxy-terminal serine-rich TRs that vary in number and amino acid sequence among different isolates of each species (7, 38). 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).

Defining the molecular characteristics of ehrlichial immunodeterminants involved in eliciting humoral immunity during infections is important for understanding the molecular basis of immunity to Ehrlichia species. Little is known regarding VLPT cellular location or function, the molecular characteristics of the immunodeterminants, or its role in the development of protective immunity. Although E. chaffeensis VLPT appears to be immunoreactive, the native VLPT protein has not been identified, nor has the full extent of immunoreactivity been determined. In this study, we report the molecular characterization of VLPT epitopes located in acidic serine-rich nonidentical TRs and the identification and cellular localization of the native protein.


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MATERIALS AND METHODS
 
Culture and purification of ehrlichiae. E. chaffeensis (Arkansas strain) and E. canis (Jake strain) were propagated and purified by size exclusion chromatography as previously described (17, 31). The fractions containing bacteria were frozen and utilized as antigen and DNA sources.

Preparation of E. chaffeensis genomic DNA and antigens. Genomic DNA and antigens were purified from E. chaffeensis (Arkansas strain) as previously described (18).

PCR amplification of E. chaffeensis VLPT gene fragments. Oligonucleotide primers for the amplification of the E. chaffeensis VLPT gene fragments were designed manually or by using Primer Select (Lasergene v5.08; DNAStar, Madison, WI) according to the sequence in GenBank (accession number AF121232) and then were synthesized (Sigma-Genosys, Woodlands, TX) (Table 1). Seven gene fragments corresponding to the four single VLPT TRs (VLPT-R4, VLPT-R3, VLPT-R2, and VLPT-R1), the C terminus of VLPT (VLPT-C), the combination of repeats R3 and R2 (VLPT-R32), and the nearly full-length VLPT (VLPT-R4321-C) containing multiple repeats (R4, R3, R2, and R1) and the C terminus of the E. chaffeensis VLPT gene were amplified using a PCR HotMaster mix (Eppendorf, Westbury, NY), with E. chaffeensis (Arkansas strain) genomic DNA as the template (Tables 1 and 2). The thermal cycling profile was as follows: 95°C for 4 min; 35 cycles of 94°C for 30 s, the annealing temperature (3°C less than the lowest primer melting temperature) for 30 s, and 72°C for the appropriate extension time (30 s/500 base pairs); a 72°C extension for 7 min; and a 4°C hold.


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  		TABLE 1. Oligonucleotide primers for amplification of E. chaffeensis VLPT gene fragments


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  		TABLE 2. Summary of E. chaffeensis VLPT recombinant polypeptide fragment characteristics

Expression and purification of recombinant E. chaffeensis VLPT proteins. The amplified PCR products were cloned directly into the pBAD/Thio-TOPO (Invitrogen, Carlsbad, CA) or pTriEx-6 3C/LIC (Novagen, Madison, WI) expression vector. Escherichia coli cells (TOP10; Invitrogen) were transformed with the plasmids containing the E. chaffeensis VLPT gene fragments, and positive transformants were screened by PCR for the presence of the insert and proper orientation and were sequenced with an ABI Prism 377XL DNA sequencer (Applied Biosystems, Foster City, CA) at the University of Texas Medical Branch Protein Chemistry Core Laboratory. Recombinant protein expression was performed for 4 h after induction with 0.2% arabinose (pBAD/Thio-TOPO) or 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) (pTriEx-6 3C/LIC). Recombinant proteins were purified under native conditions, using HisSelect columns (for pBAD/Thio-TOPO; Sigma, St. Louis, MO) or StrepTactin Superflow columns (for pTriEx-6 3C/LIC; Novagen), and quantitated with the bicinchoninic acid protein assay (Pierce, Rockford, IL) according to the manufacturer's instructions.

E. chaffeensis VLPT synthetic peptides. Five synthetic peptides, corresponding to the N-terminal fragment (VLPT-N; 17 amino acids) and four individual TR units (R4, R3, R2, and R1; 30 amino acids each) of the E. chaffeensis VLPT protein, as well as seven overlapping peptides corresponding to the different regions of R3 (R3-1 to R3-7) and a 20-amino-acid N-terminal peptide of R4 (R4-N), were synthesized (Bio-Synthesis, Lewisville, TX) (Table 3). The lyophilized powder was resuspended in molecular biology-grade water (1 mg/ml).


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  		TABLE 3. E. chaffeensis VLPT synthetic polypeptides

Antisera. A convalescent-phase anti-E. chaffeensis dog serum was derived from an experimentally infected dog (no. 2251). Sera from HME patients were a kind gift from Focus Technologies (Cypress, CA). Rabbit anti-VLPT-R3 antiserum was generated against the synthetic E. chaffeensis VLPT-R3 keyhole limpet hemocyanin-conjugated peptide by a commercial vendor (Bio-Synthesis).

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 (15), except that primary dog and human sera were diluted 1:100 and rabbit anti-VLPT-R3 antiserum was diluted 1:2,000.

Carbohydrate detection. Glycan detection on the recombinant protein VLPT was performed with a digoxigenin glycan detection kit (Roche, Indianapolis, IN), as previously described (21).

ELISA. Enzyme-linked immunosorbent assay (ELISA) plates (MaxiSorp; Nunc, Roskilde, Denmark) were coated (0.5 µg/well; 50 µl) with recombinant proteins or synthetic peptides in phosphate-buffered saline (pH 7.4). Proteins and peptides were adsorbed to the ELISA plates overnight at 4°C with gentle agitation, subsequently washed thrice with 200 µl Tris-buffered saline containing 0.2% Tween 20 (TBST) and blocked with 100 µl 3% bovine serum albumin (BSA) in TBST for 1 h at room temperature with agitation, and washed again. Convalescent-phase anti-E. chaffeensis dog or human sera diluted (1:100) in 3% BSA-TBST were added to each well (50 µl) and incubated at room temperature for 1 h with gentle agitation. The plates were washed four times, and 50 µl alkaline phosphatase-labeled goat anti-dog or -human immunoglobulin G (heavy plus light chains) secondary antibody (Kirkegaard & Perry Laboratories, Gaithersburg, MD) diluted (1:5,000) in 3% BSA-TBST was added and incubated for 1 h at room temperature. The plates were washed four times, and substrate (100 µl of BluePhos; Kirkegaard & Perry Laboratories) was added to each well. The plates were incubated for 30 min in the dark with agitation, color development was read on a microplate reader (VersaMax; Molecular Devices, Sunnyvale, CA) at A650, and data were analyzed by SoftmaxPro v4.0 (Molecular Devices). Optical density (OD) readings represent the means for three wells (± standard deviations), with the OD of the buffer-only wells subtracted.

Immunoelectron microscopy. Immunogold electron microscopy was performed on E. chaffeensis-infected DH82 cells as previously described (8), except that primary rabbit anti-VLPT-R3 peptide serum was diluted 1:10,000. Uninfected DH82 cells were used as a negative control.

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.

Analysis of secreted VLPT protein. E. chaffeensis-infected DH82 cell culture supernatants (1 ml) were collected every day without disturbing the cell monolayer and were centrifuged at high speed (10,000 x g for 5 min) to pellet cells and bacteria. Supernatants were subsequently concentrated 10-fold (Centricon Ultra centrifugal filter with a 10-kDa cutoff; Millipore, Billerica, MA) for gel electrophoresis and Western immunoblotting using an anti-VLPT-R3-specific polyclonal antibody.

Sequence analysis. The E. chaffeensis VLPT protein was evaluated for potential O-linked glycosylation and phosphorylation with the computational algorithms YinOYang v1.2 (http://www.cbs.dtu.dk/services/YinOYang) (13) and NetPhos v2.0 (http://www.cbs.dtu.dk/services/NetPhos) (3). Potential signal sequences or nonclassical secretion was identified with the computational algorithms SignalP 3.0 (http://www.cbs.dtu.dk/services/SignalP) (2) and SecretomeP 2.0 (http://www.cbs.dtu.dk/services/SecretomeP) (1) trained on gram-negative bacteria. Nucleic acid and amino acid alignments were performed with MegAlign (Lasergene v5.08; DNAStar). E. chaffeensis VLPT epitopes were examined for homology to other Ehrlichia sp. proteins (including VLPT orthologs) by using the protein-protein basic local alignment search tool (BLAST [http://www.ncbi.nlm.nih.gov/BLAST]).


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RESULTS
 
E. chaffeensis VLPT protein composition and characteristics. Serine (33 residues; 16.7%), leucine (22 residues; 11.1%), glutamate (20 residues; 10.1%), and aspartate (17 residues; 8.6%) were the most frequently occurring amino acids in the E. chaffeensis VLPT protein, accounting for 46.5% of the entire amino acid content (Fig. 1A). Moreover, in the repeat region of the VLPT protein, the occurrences of these four residues became more frequent, with a composition of 20%, 12.5%, 13.3%, and 10%, respectively, which accounted for 55.8% of the entire repeat region amino acid content. Four cysteine residues associated with disulfide bonds were present in the carboxyl-terminal domain of the protein, but this domain was dominated by tyrosine residues (19.7%). Due to the large proportion of the strongly acidic residues glutamate and aspartate, the VLPT protein was highly acidic (pI 3.8). The N-terminal region (17 amino acids) and the largest domain, the TR region (120 amino acids), were highly acidic (pI 3.2 and 3.8, respectively), and the carboxyl-terminal domain (61 amino acids) was the least acidic domain (pI 4.7).


Figure 1
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  		FIG. 1. (A) Amino acid sequence of VLPT protein showing all domains and locations of four TRs (numbers of amino acids are given in parentheses). (B) Phylogenetic tree showing the relationships of the four E. chaffeensis VLPT repeats. The scale represents the amino acid percent identity.

The TRs of VLPT were nonidentical, but R3 and R2 had the highest amino acid identity (83%) and R4 and R1 had 53% and 49% identity with R3, respectively (Fig. 1B). A BLAST search with amino acid sequences from VLPT-R3 and VLPT-R4 found no homology between the VLPT repeats and other known ehrlichial proteins or proteins from organisms in closely related genera.

Identification of native VLPT protein. Western blotting identified a native protein with a molecular mass of ~32 kDa (~6.2 kDa larger than the predicted mass of 25.8 kDa) and five less prominent proteins (22 to 30 kDa) in E. chaffeensis whole-cell lysates and supernatants from E. chaffeensis-infected cells that reacted with monospecific rabbit antiserum against the synthetic VLPT-R3 peptide (Fig. 2A). Furthermore, anti-VLPT-R3 antibody did not react with E. canis whole-cell lysate (Fig. 2A). A protein of similar size (~32 kDa) in E. chaffeensis whole-cell lysates and supernatants from infected cultures also reacted with anti-E. chaffeensis dog serum (Fig. 2B). Preimmunization rabbit serum or dog serum controls did not recognize E. chaffeensis whole-cell lysates or supernatants (data not shown).


Figure 2
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  		FIG. 2. Identification of native VLPT in E. chaffeensis whole-cell lysates (lane 1), supernatants derived from E. chaffeensis-infected cells (lane 2), and E. canis whole-cell lysates (lane 3) incubated with anti-VLPT-R3 peptide antibody (A) and anti-E. chaffeensis dog serum (B). Preimmunization rabbit serum or dog serum controls did not recognize E. chaffeensis whole-cell lysates or supernatants (data not shown).

Epitope-containing regions of VLPT. To determine the major epitope-containing regions of VLPT, recombinant proteins corresponding to distinct VLPT domains (R4321-C, R32, R1, R2, R3, R4, and C) (Fig. 3) were expressed. All of the recombinant VLPT proteins expressed in pBAD/Thio-TOPO exhibited molecular masses that were substantially larger by SDS-PAGE than those predicted by amino acid sequence, except for VLPT-R3 and VLPT-C (Table 2). VLPT-R32 expressed in an alternative vector, pTriEx6 3C/LIC, with a substantially smaller N-terminal fusion protein (2.4 kDa compared to 13.1 kDa for pBAD/Thio) also exhibited an increased molecular mass (3.7 kDa larger than predicted), but it was smaller than that of VLPT-R32 expressed in pBAD/Thio (5.2 kDa larger than predicted). The increased molecular masses exhibited by recombinant VLPT proteins suggested that the protein was posttranslationally modified; moreover, several predicted glycosylation sites were identified on VLPT by a computational algorithm. However, carbohydrate was not detected on any recombinant E. chaffeensis VLPT polypeptides (data not shown). To further confirm the actual molecular mass of one recombinant VLPT protein, the mass of the two-repeat-containing VLPT-R32 expressed in pTriEx6 3C/LIC was determined by MALDI-TOF. The mass of VLPT-R32 was 9,206 Da, which was slightly smaller than the mass (9,325 Da) predicted by the amino acid sequence (excluding the 2.4-kDa expression tag), demonstrating that posttranslational modifications were not present.


Figure 3
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  		FIG. 3. Schematic of synthetic and recombinant peptides used to map the VLPT epitopes.

By Western immunoblotting, the large TR-containing proteins R4321-C and R32 and individual repeat units R2 and R3 reacted strongly with anti-E. chaffeensis dog serum, but recombinant fragments representing the carboxy-terminal domain (C) and repeat units R1 and R4 were not reactive with anti-E. chaffeensis dog serum (Fig. 4A). The reactivity of VLPT (synthetic polypeptides and corresponding recombinant proteins) with anti-E. chaffeensis dog serum was also examined by ELISA to identify potential conformational epitopes (Fig. 4B). VLPT-N (synthetic), VLPT-C (recombinant), and VLPT-R1 (synthetic and recombinant) polypeptides did not react with anti-E. chaffeensis dog serum. Similar to results by Western immunoblotting, VLPT-R3 and -R2 peptides (synthetic and recombinant) reacted strongly with anti-E. chaffeensis dog serum; however, VLPT-R3 (synthetic) was more immunoreactive than VLPT-R2 (synthetic). Conversely, recombinant VLPT-R4, which was not immunoreactive by Western immunoblotting, reacted (synthetic and recombinant) strongly with anti-E. chaffeensis dog serum by ELISA, indicating that a conformational epitope was present in VLPT-R4 (Fig. 4A and B).


Figure 4
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  		FIG. 4. Immunoreactivities of synthetic and recombinant peptides of E. chaffeensis VLPT with anti-E. chaffeensis dog (no. 2251) serum. (A) SDS-PAGE and total protein staining of purified recombinant peptides (top) and corresponding Western immunoblot probed with anti-E. chaffeensis dog serum (bottom). M, Precision protein standard (Bio-Rad); Ctrl, purified recombinant thioredoxin. (B) Immunoreactivities by ELISA of small recombinant and corresponding synthetic VLPT polypeptides (N [synthetic only], R1, R2, R3, and R4) and large VLPT protein fragments (recombinant only; C, R4321-C, and R32). The OD readings represent the means for three wells (± standard deviations), with the OD of the buffer-only wells subtracted.

Identification of major VLPT immunodeterminants. To further localize the major epitopes of the E. chaffeensis VLPT protein, seven overlapping peptides (designated R3-1 to R3-7) corresponding to the different locations within VLPT-R3 were incubated with anti-E. chaffeensis dog serum (Fig. 3 and 5A). Peptides R3-6 and R3-7 (C-terminal region) were not immunoreactive, but R3-2, R3-3, R3-4, and R3-5, corresponding to the N-terminal region, were found to react similarly and strongly with anti-E. chaffeensis dog serum by ELISA (Fig. 4B), indicating that the N-terminal region (23 amino acids; SDLHGSFSVELFDPFKEAVQLGN) of VLPT-R3 contained a major antibody epitope. Peptide R3-3 (14 amino acids; HGSFSVELFDPFKE) was the smallest peptide that reacted strongly with anti-E. chaffeensis dog serum (Fig. 5A and B). Peptides R3-1 and R3-6, which differed by three (C-terminal) and five (N-terminal) amino acids, respectively, were not reactive (Fig. 5A and B).


Figure 5
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  		FIG. 5. (A) Sequence and orientation of overlapping peptides (seven peptides) representing VLPT-R3. (B) Immunoreactivities of overlapping VLPT-R3 peptides by ELISA with anti-E. chaffeensis dog serum.

To examine and compare the immunodeterminant in VLPT-R4, a 20-amino-acid peptide (HEPSHLELPSLSEEVIQLES) corresponding to R3-5 in VLPT-R3 (Fig. 4A) was not immunoreactive with either dog serum or patient sera (data not shown), indicating that the third epitope of VLPT in R4 was molecularly distinct, consistent with the divergence noted in the amino acid sequences of R4 compared to R3 and R2 (Fig. 1B).

Immunoreactivity of VLPT-R3 peptides with HME patient sera. Three HME patient sera (patient no. 1, 4, and 12) that had detectable E. chaffeensis antibodies by immunofluorescence assay were used to examine the immunoreactivity of VLPT-R4, -R3, and -R2 (synthetic and recombinant) by ELISA (Fig. 6A to C, respectively). Consistent with the immunoreactivity exhibited with anti-E. chaffeensis dog serum, VLPT-R3 and -R2 also exhibited the strongest immunoreactivity with HME patient sera, and two patients (no. 1 and 12) exhibited a strong antibody response to VLPT-R4 (Fig. 6A to C).


Figure 6
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  		FIG. 6. (A to C) Immunoreactivities of synthetic and recombinant E. chaffeensis VLPT repeats (R2, R3, and R4) with three HME patient sera by ELISA. Ctrl, purified recombinant thioredoxin. (D) Synthetic E. chaffeensis VLPT-R3 reacting in ELISA with 14 HME patient sera (lanes 1 to 14), anti-E. chaffeensis dog serum (lane 15), and normal human serum (lane 16). The normal human serum did not recognize other peptides and proteins as well (data not shown).

The immunoreactivities of the three HME patient sera with the seven overlapping synthetic peptides (R3-1 to R3-7) from VLPT-R3 were determined by ELISA (Fig. 6A to C). Peptides R3-4 (17 amino acids) and R3-5 (20 amino acids), which contained similar amino acid sequences (Fig. 5A), reacted strongly and consistently with all HME patient sera tested (Fig. 6A to C). Comparing the overlapping peptides, the minimum peptide sequence critical for this immunodeterminant was 17 amino acids (peptide R3-4). Antibodies from HME patients and a dog experimentally infected with E. chaffeensis reacted similarly to VLPT-R3 (Fig. 4B and 6A to C). However, antibodies in human sera were directed primarily against peptides R3-4 and R3-5 within VLPT-R3 (Fig. 6A to C). Normal human serum did not recognize these peptides and proteins (data not shown).

The reactivity of VLPT-R3 with a larger panel of HME patient sera (from 14 patients) that had detectable E. chaffeensis antibodies was determined. All patient sera reacted with VLPT-R3 (synthetic) (Fig. 6D), indicating that this epitope is consistently recognized by humans, and the reactivity of antibodies in patient sera with this epitope completely correlated with immunofluorescence assay results. Normal human serum did not recognize VLPT-R3 (Fig. 6D, lane 16).

Temporal secretion of E. chaffeensis VLPT. VLPT was detected in supernatants from infected cells as early as 1 day postinfection and increased in quantity through 6 days postinfection (Fig. 7). The VLPT protein was not observed in uninfected DH82 cell culture supernatant.


Figure 7
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  		FIG. 7. Western immunoblot of DH82 cell culture supernatant (0 to 6 days postinfection; lanes 1 to 7, respectively) of E. chaffeensis probed with VLPT-R3 peptide antibody. M, Precision protein standard (Bio-Rad).

Cellular and extracellular localization of VLPT. Several characterized ehrlichial proteins are differentially expressed on dense-core ehrlichiae (gp120, gp36, and gp47). However, like its ortholog gp19 of E. canis, the E. chaffeensis VLPT protein was observed on the membranes of morulae and the surfaces of both reticulate and dense-core ehrlichiae but was also detected on the morula fibrillar matrix by immunoelectron microscopy (Fig. 8A). Anti-VLPT-R3 antibody did not react with uninfected DH82 cells (Fig. 8B).


Figure 8
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  		FIG. 8. Electron photomicrograph of an ultrathin section of E. chaffeensis-infected DH82 cells demonstrating E. chaffeensis VLPT localization in both reticulate and dense-core ehrlichiae (A) and a corresponding ultrathin section containing uninfected DH82 cells (negative control) (B). Cells in both panels were incubated with rabbit VLPT-R3 peptide antibody (1:10,000). Bar = 1 µm.


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DISCUSSION
 
The initial description of the E. chaffeensis VLPT gene focused on the applications of the gene for molecular diagnostics and epidemiology. Hence, the VLPT gene has been utilized frequently to differentiate isolates based on differences in the number of TR units and sequence variation present in the gene (32, 35). Although a previous study demonstrated that recombinant VLPT reacted with antibodies in HME patient sera, the immunologic properties of the VLPT protein were not fully defined (32). Notably, the VLPT protein has never been identified conclusively in E. chaffeensis native whole-cell lysates, and major immunoreactive proteins corresponding to its reported molecular mass of 44 kDa (double the predicted size) have never been identified. 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. The role of the E. chaffeensis VLPT protein in ehrlichial pathobiology is also unknown, and its lack of relationship with other known bacterial proteins provides no clues regarding its potential function. A remarkable feature of VLPT and E. canis gp19 is the homologous carboxy-terminal domain dominated by tyrosine, suggesting that it is a functionally important conserved domain.

The discrepancy in the apparent molecular mass of the E. chaffeensis VLPT protein (Arkansas strain) observed in this study (~32 kDa) and that of the recombinant VLPT protein (~44 kDa) reported in a previous study (32) was noted, but the reasons behind this difference are not clear. To further confound this issue, native VLPT was never identified in the previous study. Nevertheless, the mass of the native VLPT protein (~32 kDa) identified from the ehrlichial lysate by anti-VLPT-R3 antibody and the mass of our recombinant VLPT protein (without fusion tag) were in agreement. Hence, the evidence generated in this study indicates that the mass of the VLPT protein (recombinant and native) is ~32 kDa, which is larger than the predicted mass (22.4 kDa) but substantially smaller than that previously reported (32).

Four pairs (gp200s, gp120/gp140, gp47/gp36, and VLPT/gp19) of major immunoreactive protein orthologs in E. chaffeensis and E. canis have been identified. Two ortholog pairs are TR-containing proteins, and VLPT and gp19 also appear to be similar. Although E. canis gp19 lacks multiple repeats found in E. chaffeensis VLPT, it has a Ser/Thr/Glu-rich patch that is similar in size and composition to that of a single serine-rich repeat unit of VLPT, and the major immunodeterminant of gp19 was mapped to the STE-rich patch. Similarly, antibody epitopes have been identified in other serine-rich TR-containing ehrlichial protein orthologs, including gp36/47 and gp120/140 (7, 37).

Except for p28/p30, all of the major immunoreactive proteins of E. chaffeensis and E. canis that have been characterized are highly acidic due to a predominance of glutamate and aspartate, but they also have a large proportion of polar amino acids, such as serine, which are present at a higher frequency within TRs found in these proteins (7, 16, 21, 38, 39). Moreover, major antibody epitopes of these proteins have been mapped to these serine-rich acidic TRs or acidic domains (7, 16, 21, 24, 38, 39). The amino acid composition of E. canis gp19 consisted predominantly of three amino acids, i.e., serine, glutamate, and aspartate. Consistent with other major immunoreactive proteins, including gp19, VLPT has a similar predominance of serine, glutamate, and aspartate, which are more pronounced in the TR region. The association of the polar and acidic amino acids is not well understood, but the high frequency of them suggests a direct relationship between the host immune response and acidic serine-rich repetitive sequences and domains.

We previously reported detection of carbohydrates on recombinant ehrlichial TR-containing proteins that exhibited larger-than-predicted masses similar to those of their native counterparts. Furthermore, VLPT has been reported to exhibit a larger-than-predicted mass by gel electrophoresis, a finding that was also observed in this study with both native and recombinant VLPT proteins (32). Thus, we considered the possibility that glycosylation was responsible for this difference. Serine and threonine residues are linkage sites for O-glycans, and some of these amino acids were predicted to be glycan attachment sites on the VLPT. However, unlike other ehrlichial proteins, we could not detect carbohydrate on the VLPT, and the mass (as determined by MALDI-TOF) of a recombinant two-repeat-containing fragment (VLPT-R32) was consistent with its predicted mass, confirming that the abnormal migration was not due to posttranslational modification of VLPT tandem repeats. One potential explanation for the increased electrophoretic mobility is the fact that VLPT is a highly acidic protein. Others have reported that highly acidic proteins, such as ribonuclease U2 and caldesmon, exhibit anomalous electrophoretic behavior that could be normalized after neutralization (11, 12, 22). The high acidic amino acid content and low overall pI (3.8) of VLPT are a likely explanation for its electrophoretic behavior and may also contribute to the anomalous behavior of other highly acidic TR-containing ehrlichial proteins.

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). Thus, antibodies directed against R3 would likely cross-react with R2. Therefore, the R3 epitope appears to be the primary immunodeterminant for both human and dog anti-VLPT antibodies. Interestingly, the R3 immunodeterminant appeared to be highly dependent on three terminal amino acids (AVQ) in peptide R3-4 when detected by human antibodies, whereas the antibodies reactive in canine serum appeared to be more dependent on three amino acids (FKE) directly upstream. Thus, R3-3 is the minimum epitope sequence (14 amino acids) for recognition by dog antibodies, and R3-4 (17 amino acids), containing three additional C-terminal amino acids, is essential for antibody reactivity with human sera. Interestingly, R4, the most divergent repeat, was not reactive by Western immunoblotting but was reactive with antibody in ELISA, suggesting that a distinct conformational epitope was present in R4. Conformational epitopes have been described for Ehrlichia and Anaplasma species (6, 23). Thus, R4 contributes to the immunoreactivity of VLPT independent of R3. The smaller R4 peptide (20 amino acids) that corresponds to R3-5 in VLPT-R3 was not immunoreactive; however, the full repeat (30 amino acids) was immunoreactive, which supports our conclusion that this epitope is discontinuous and requires the entire repeat sequence to create the epitope.

The epitopes identified in VLPT repeat units appear to be species specific, as the anti-VLPT-R3 antibody did not cross-react with the closely related E. canis and amino acid homology was not observed between VLPT-R2, -R3, and -R4 and proteins of other Ehrlichia species or closely related pathogens. 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). The current findings further support our previous conclusion that antibodies generated against E. chaffeensis are directed primarily at species-specific epitopes. Hence, antibodies generated against one Ehrlichia species may provide little or no protection against a closely related pathogen, such as E. canis in this case. However, species-specific antigens such as VLPT are excellent candidates for the development of sensitive species-specific immunodiagnostics and are useful for epidemiologic studies.

There is evidence that ehrlichial TR-containing proteins such as E. chaffeensis gp120 and gp47 are secreted (7, 29). In this study, we demonstrate that the VLPT protein is also secreted. The mechanism of secretion appears to be sec independent because VLPT does not have an amino-terminal signal sequence. VLPT was predicted by SecretomeP 2.0 to be secreted by a nonclassical and leaderless secretion system; therefore, secretion of VLPT and other TR-containing proteins may occur by a similar mechanism to that for E. chaffeensis gp120 and gp47, which also lack an N-terminal signal sequence but are found outside the bacterium in the morula and in infected cell culture supernatants. Genes encoding type IV secretion system components have been reported for both Ehrlichia and Anaplasma (9, 26), and AnkA of Anaplasma phagocytophilum appears to be secreted by this system (14). However, the VLPT does not appear to contain a type IV effector protein consensus sequence (R-X7-R-X-R-X-R-X-Xn) and could be a substrate of other secretion systems (sec dependent and sec independent) that have been identified in Ehrlichia species (9).

Distinct from the differential expression (on dense-core ehrlichiae) of E. chaffeensis gp120 and gp47, but consistent with the localization of E. canis gp19 (7, 29), E. chaffeensis VLPT protein was detected on both morphological forms, i.e., reticulate and dense-core ehrlichiae, but was primarily found extracellularly, associated with the morula fibrils and morula membrane. Thus, the VLPT protein does not appear to be a major surface protein and is not associated specifically with the infectious form of ehrlichiae (dense-cored). The secretion of VLPT into the morula space and membrane indicates a potentially important role in morula maintenance or as a virulence factor.

The majority of the characterized major immunoreactive proteins of Ehrlichia species are acidic TR-containing proteins that have common amino acid usage and elicit strong humoral immune responses directed at TRs. The host immune response appears to be directed primarily at epitopes within TRs, which suggests that all of these proteins interact similarly with the host immune response. Future studies will determine whether antibodies directed at specific epitopes in TR proteins are protective, and other studies may provide insight into the function of TR proteins in ehrlichial pathobiology.


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ACKNOWLEDGMENTS
 
This work was supported by National Institutes of Health grant R01 AI 071145-01 (to J.W.M.), the Clayton Foundation for Research, and the UTMB Sealy Center for Vaccine Development.

We thank Xue-jie Yu and David H. Walker for reviewing the manuscript and providing helpful suggestions.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Pathology, University of Texas Medical Branch, Galveston, TX 77555-0609. Phone: (409) 747-2498. Fax: (409) 747-2455. E-mail: jemcbrid{at}utmb.edu Back

{triangledown} Published ahead of print on 22 January 2008. Back

Editor: R. P. Morrison


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Infection and Immunity, April 2008, p. 1572-1580, Vol. 76, No. 4
0019-9567/08/$08.00+0     doi:10.1128/IAI.01466-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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