Two Monoclonal Antibodies with Defined Epitopes of P44 Major Surface Proteins Neutralize Anaplasma phagocytophilum by Distinct Mechanisms
http://www.100md.com
感染与免疫杂志 2006年第3期
Department of Veterinary Biosciences, College of Veterinary Medicine, Ohio State University, Columbus, Ohio 43210
ABSTRACT
Anaplasma phagocytophilum is an obligatory intracellular bacterium that causes human granulocytic anaplasmosis. The polymorphic 44-kDa major outer membrane proteins of A. phagocytophilum are dominant antigens recognized by patients and infected animals. However, the ability of anti-P44 antibody to neutralize the infection has been unclear due to a mixture of P44 proteins with diverse hypervariable region amino acid sequences expressed by a given bacterial population and lack of epitope-defined antibodies. Monoclonal antibodies (MAbs) 5C11 and 3E65 are directed to different domains of P44 proteins, the N-terminal conserved region and P44-18 central hypervariable region, respectively. Passive immunization with either MAb 5C11 or 3E65 partially protects mice from infection with A. phagocytophilum. In the present study, we demonstrated that the two monoclonal antibodies recognize bacterial surface-exposed epitopes of naturally folded P44 proteins and mapped these epitopes to specific peptide sequences. The two MAbs almost completely blocked the infection of the A. phagocytophilum population that predominantly expressed P44-18 in HL-60 cells by distinct mechanisms: MAb 5C11 blocked the binding, but MAb 3E65 did not block binding or internalization. Instead, MAb 3E65 inhibited internalized A. phagocytophilum to develop into microcolonies called morulae. Some plasma from experimentally infected horses and mice reacted with these two epitopes. Taken together, these data indicate the presence of at least two distinct bacterial surface-exposed neutralization epitopes in P44 proteins. The results indicate that antibodies directed to certain epitopes of P44 proteins have a critical role in inhibiting A. phagocytophilum infection of host cells.
INTRODUCTION
Human granulocytic anaplasmosis (formerly human granulocytic ehrlichiosis) is an emerging tick-borne zoonosis that has been reported in the United States and Europe (2, 27, 33). Human granulocytic anaplasmosis is caused by infection of an obligatory intracellular bacterium, Anaplasma phagocytophilum, in the family Anaplasmataceae. This bacterium lacks pili, a capsule (28), lipopolysaccharides, and peptidoglycans (18), suggesting that the outer membrane proteins play an important role in its interaction with host granulocytes. A family of proteins with molecular sizes of the 44-kDa range (P44s, also called Msp2s) was shown to be present in the isolated outer membrane fraction of A. phagocytophilum by Western blot analysis and on the surface of A. phagocytophilum within the inclusion by immunogold labeling in the postembedded electron microscopy specimens (14).
P44 proteins are encoded by the p44 (msp2) polymorphic multigene family. Despite its small genome size (1.47 Mb) due to the ongoing reductive genome evolution among members of the family Anaplasmataceae, the A. phagocytophilum genome contains approximately 90 p44 paralogues, suggesting that this large expansion of p44 paralogues has given A. phagocytophilum a survival advantage, perhaps by allowing it to escape host immunoclearance. P44 proteins consist of a single central hypervariable region of approximately 94 amino acid residues, an N-terminal conserved region of approximately 186 amino acids, and a C-terminal conserved region of approximately 146 amino acids; the N- and C-terminal regions flank the central hypervariable region (21, 36). There are three short conserved segments including absolutely conserved two cysteines within the hypervariable region of all predicted P44 proteins (21).
Infected animals develop antibodies directed against the N-terminal conserved region as well as against the hypervariable region (14, 34, 38). P44s undergo antigenic variation during infection in human granulocytic anaplasmosis patients and in experimentally infected horses (3, 34). The hypervariable region of P44 molecules has been assumed to be exposed on the bacterial surface and involved in antigenic variation and immune evasion (3, 14, 21, 34, 36). However, since epitopes of anti-P44 antibodies have never been defined, whether or which part of the hypervariable region or any other regions of naturally folded P44 molecules is exposed to the surface of the intact bacterium has been unknown.
Human granulocytic anaplasmosis patients, unless immunocompromised, generally develop antibodies to P44s; thus, P44s are considered useful antigens for serological diagnosis of human granulocytic anaplasmosis (12, 21, 22, 32, 37). Horses and mice experimentally infected with A. phagocytophilum also develop an antibody to P44s (13, 14, 34). It is less clear whether antibodies to P44s are protective from infection. Ijdo et al. (11) reported lack of protection on day 15 postchallenge in mice immunized with a recombinant P44 protein. Two anti-Msp2 (P44) monoclonal antibodies (MAbs) and a recombinant Msp2 only weakly block A. phagocytophilum binding and infection of HL-60 cells (26). The passive immunization of nave mice with MAbs directed against P44s partially protects mice from infection (14). The results of these studies have given an overall impression that antibodies to directed P44 (Msp2) do not have a significant role in immunoprotection.
However, the previous studies defined neither epitopes of the MAbs or the epitopes of antibodies developed by immunization with the recombinant P44 protein nor p44 species predominantly expressed by the A. phagocytophilum population used to infect the mice or HL-60 cells. Thus, it is unclear whether this poor protection in mice or HL-60 cells is simply due to (i) poor neutralization ability of particular anti-P44 antibodies involved, (ii) lack of surface exposure of the target epitope on the intact bacteria, or (iii) epitope mismatch between anti-P44 antibodies and P44 proteins expressed by the organisms used for infection.
Our MAb 3E65 obtained through screening by immunofluorescence followed by Western blot analysis (14) recognizes a linear epitope within the recombinant hypervariable region of P44-18 protein (33). MAb 5C11 reacts with a linear epitope within the recombinant partial P44-1 protein, which consists of most of the conserved N-terminal region and a part of the hypervariable region of P44-1 (14, 37), with the A. phagocytophilum HZ strain cultured in HL-60 cells at 37°C, which expresses various p44s, but not p44-1 (36), and with diverse P44s derived from several other strains of A. phagocytophilum so far examined (14). Thus, the MAb 5C11 epitope has been considered to be within the conserved P44 N terminus, but not within the hypervariable region of P44-1.
Passive immunization with MAbs 5C11 and 3E65 partially protects nave mice from infection with A. phagocytophilum HZ (14), indicating that P44 proteins contain at least two in vivo neutralizable B-cell epitopes. In the present study, we defined the two neutralization sites on P44 molecules by epitope peptide mapping and used the MAbs to delineate their bacterial surface exposure and inhibitory mechanisms of infection of host cells. The present results support the idea that large expansion of p44 paralogues allows A. phagocytophilum to escape neutralizing antibodies.
MATERIALS AND METHODS
Bacteria. A. phagocytophilum HZ strain isolated from a human granulocytic anaplasmosis patient in 1995 (29) and Ehrlichia chaffeensis Arkansas were cultured in HL-60 cells (a human promyelocytic leukemia cell line) (American Type Culture Collection, Manassas, VA) in RPMI 1640 medium (Gibco-BRL, Grand Island, NY) supplemented with 5% fetal bovine serum (FBS; Bio-technologies Inc., Parker Ford, PA), and 2 mM L-glutamine (Gibco-BRL). Cell cultures were incubated at 37°C in a humidified 5% CO2-95% air atmosphere. The percentage of infected cells was determined by Diff-Quik staining (Baxter Scientific Products, Obetz, OH).
Cloning and expression of rP44N and rP44N-N. Primer pairs TCCGAATTCGACTGGTGGTGCGGGAT and CGCAAGCTTCTAATCAGTCTGCCCTCCGAATTCGACTGGTGGTGCGGGAT and CGCAAGCTTCTACTTAAACCCAATCCGA were designed to clone the DNA fragments encoding recombinant P44N (rP44N; P44N terminal from position 35 to 162, 128 amino acids) and recombinant P44N-N (rP44N-N; N-terminal portion of rP44N, from positions 35 to 98, 64 amino acids) based on the full-length P44-18 sequence at the p44 expression locus (36). The EcoRI and HindIII sites were underlined. PCR products of the expected sizes were obtained and digested with EcoRI and HindIII. These fragments were then ligated into the EcoRI and HindIII sites of the pET33b(+) vector (Novagen Inc., Madison, WI). The resulting plasmid was amplified in Escherichia coli Novablue cells (Novagen). Escherichia coli BL21(DE3) cells (Novagen) were transformed with the recombinant plasmid and induced to express rP44N and rP44N-N with isopropyl-thio--D-galactoside. The recombinant proteins include 41 amino acids at the N terminus derived from the pET33b(+) vector. The rP44N and rP44N-N inclusion bodies were purified using the B-PER II bacterial protein extraction reagent (Pierce, Rockford, IL). The recombinant proteins were then Ni-affinity purified with a His-Select cartridge (Sigma, St. Louis, MO).
Surface labeling of host cell-free A. phagocytophilum with MAb 5C11 and 3E65. Host cell-free organisms were isolated as previously described (35) and cytocentrifuged on glass slides. Organisms were fixed with 2% paraformaldehyde in phosphate-buffered saline (PBS; 137 mM NaCl, 10 mM Na2HPO4, 2.7 mM KCl, 1.8 mM KH2PO4; pH 7.2) for 1 h at room temperature and then incubated with 5C11, 3E65, or normal mouse immunoglobulin G (IgG) only or with both 5C11 and a rabbit antibody directed against the recombinant A. phagocytophilum NtrX transcription factor (T.-H. Lai, Y. Kumagai, and Y. Rikihisa, unpublished data) for 1 h at 37°C. After being washed twice with PBS, cells were incubated with Alexa Fluor 555-conjugated (red fluorescence) goat anti-mouse IgG antibody (Molecular Probes, Eugene, OR) or with both Alexa Fluor 555-conjugated goat anti-rabbit IgG and Alexa Fluor 488-conjugated (green fluorescence) goat anti-mouse IgG antibody (Molecular Probes) for 1 h at 37°C. As the control, host cell-free organisms were fixed with methanol at –20°C for 5 min to permeabilize the organism membrane and then double labeled with both MAb 5C11 and anti-NtrX, with both MAb 3E65 and anti-NtrX, or with both MAb 5C11 and rabbit preimmune serum for 1 h at 37°C. After being washed twice with PBS, cells were incubated with both Alexa Fluor 555-conjugated goat anti-rabbit IgG and Alexa Fluor 488-conjugated goat anti-mouse IgG antibody for 1 h at 37°C. Bacteria were then washed and observed under an Eclipse E400 epifluorescence microscope with a xenon–mercury light source (Nikon, Melville, NY).
Effects of P44 MAbs on binding, internalization, and infection in vitro. For the binding analysis, the host cell-free A. phagocytophilum was freshly prepared from 2.5 x 104 A. phagocytophilum-infected HL-60 cells (>90% cells infected) as previously described (35). The preparation was then incubated with MAbs 5C11 (IgG2b), 3E65 (IgG1) (14), or normal mouse IgG (Sigma)-nonrelated mouse MAbs with the isotype of IgG2b (hybridoma culture supernatant) (final concentration, 1 mg/ml), or with RPMI 1640 medium at room temperature for 30 min with gentle shaking. Each mixture was then added to 2 x 105 uninfected HL-60 cells (final concentration, 1.3 x 106 cells/ml) in RPMI 1640 medium supplemented with 5% FBS and 2 mM L-glutamine. After incubation at room temperature for 15 min with shaking, each mixture was incubated at 37°C in 95% air-5% CO2 for 45 min. Cells were harvested by centrifugation at 750 x g for 5 min and washed with PBS to remove the unbound A. phagocytophilum; this washing procedure was then repeated twice. The mixture was cytocentrifuged and fixed with cold methanol for 5 min and sequentially incubated with horse anti-A. phagocytophilum plasma and Cy3-conjugated anti-horse IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). The number of bound organisms was scored in 100 HL-60 cells in triplicate samples.
For analysis of infection, the host cell-free A. phagocytophilum freshly prepared from each sample of 4 x 105 A. phagocytophilum-infected HL-60 cells (>90% cells infected) as previously described (35), was incubated with various concentration of either MAb 5C11 or 3E65 (14), nonrelated mouse MAb with the isotype of IgG2b, nonrelated mouse MAb with the isotype of IgG1, normal mouse IgG, or RPMI 1640 medium at room temperature for 30 min with gentle shaking. As a negative control, the same number of host cell-free Ehrlichia chaffeensis cells were incubated with MAb 5C11, MAb 3E65, normal mouse IgG or RPMI 1640 medium under the same conditions. Each of the mixtures was added to 2 x 105 uninfected HL-60 cells (final concentration, 106 cells/ml) in RPMI 1640 medium supplemented with 5% FBS and 2 mM L-glutamine and incubated at 37°C in 95% air-5% CO2. After 12 h, the medium was replaced with fresh RPMI 1640 medium supplemented with 5% FBS and 2 mM L-glutamine. The host cell-free organisms were then coincubated with HL-60 cells at 37°C for several days to allow the growth of internalized A. phagocytophilum (19). The infection of A. phagocytophilum and Ehrlichia chaffeensis was evaluated by Diff-Quik staining. The infectivity was scored in 100 HL-60 cells in triplicate samples.
To study the effects of MAbs on the internalization and development of A. phagocytophilum in HL-60 cells, the cell-free organisms were prepared as described above and then incubated with MAb 3E65 (final concentration, 1 mg/ml), or with RPMI 1640 medium at room temperature for 30 min with gentle shaking. Each mixture was then added to uninfected HL-60 cells as described above. After incubation at room temperature for 15 min with shaking, each mixture was incubated at 37°C for 1, 4, 16, and 32 h. At 12 h postinoculation, the medium was replaced with fresh RPMI 1640 medium supplemented with 5% FBS and 2 mM l-glutamine. Cells were harvested and washed as described above, followed by the fixation with 2% paraformaldehyde at room temperature for 1 h. The extracellular bacteria were first stained with horse anti-A. phagocytophilum plasma and Cy3-conjugated goat anti-horse IgG in the absence of saponin, whereas the total extracellular and intracellular bacteria were stained with the same antibodies in the presence of 0.3% saponin.
In order to estimate lysosomal fusion, double immunofluorescence labeling was performed using phycoerythrin-MAb against human CD63 (IgG1; BioLegend, San Diego, CA) and horse anti-A. phagocytophilum plasma (detected with fluorescein isothiocyanate-conjugated goat anti-horse IgG [Jackson ImmunoResearch]) in the presence of 0.3% saponin. All cells were washed three times with PBS to remove unbound antibodies before observation with a Nikon Eclipse E400 fluorescence microscope. Bacteria or inclusions per 100 HL-60 cells were scored in three independent experiments. Statistical analyses were performed by using analysis of variance and the Tukey honestly significant differences test or by Student's t test, and a P of <0.05 was considered significant.
Peptide synthesis and peptide-pin ELISA analysis of MAbs 5C11 and 3E65 and infected horse, mouse, and human plasma. The octamer peptide libraries were synthesized for the P44N-N region (64 amino acids) and the domain bordered by two cysteines in the P44-18 hypervariable (P44-18hv C-C) region (30 amino acids) with three and four overlapping amino acids, respectively, using noncleavable multipin synthesis technology and fluorenylmethoxycarbonyl (Fmoc) chemistry (Mimotopes Pty Ltd., Victoria, Australia) (10). After disruption of the peptide-pins with 0.1 M sodium phosphate buffer containing 1% sodium dodecyl sulfate and 0.1% 2-mercaptoethanol (pH 7.2) and washing with hot water, nonspecific binding sites of pins were blocked with 200 μl of blocking solution containing 2% (wt/vol) bovine serum albumin in PBS-Tween 20 in 96-well plates for 1 h at room temperature.
Sets of peptide-bound pins were washed once with PBS containing 0.1% Tween 20 for 10 min and then incubated with either MAb 5C11 (1:600 dilution), MAb 3E65 (1:100 dilution), or one of the following in blocking solution at 4°C overnight: a 1:50 dilution of horse preimmune plasma from EQ001, EQ005, and EQ006; plasma from seven horses from a region where human granulocytic anaplasmosis is nonendemic (Columbus, Ohio); plasma from horse EQ001 (immunofluorescence titer, 1:320) on day 16 postinoculation of A. phagocytophilum (13); plasma from horse EQ005 (immunofluorescence assay titer, 1:5,000) on day 31 after infected-tick placement; plasma from horse EQ006 (immunofluorescence titer, 1:640) on day 22 postinoculation (33); one of three pooled mouse preimmune plasma samples (for each pool, there were >5 mice); and one of three plasma samples pooled from three infected ICR, C3H/HeN, and C3H/HeJ strain mice (immunofluorescence titer, 1:1,000) on day 31 postinoculation. Immunofluorescence titers of these plasma against A. phagocytophilum were determined as previously described (14).
After being washed four times as described above, horseradish peroxidase-labeled goat anti-mouse or horse IgG (heavy and light chain) (Kirkegaard & Perry Laboratories, Gaithersburg, MD) secondary antibodies were used. All secondary antibodies were diluted 1:500 or 1:200 in 1% (vol/vol) sheep serum (Sigma). The peptide pins were placed in the wells filled with the corresponding secondary antibodies and incubated for 1 h at room temperature. Samples were washed four times, then the horseradish peroxidase substrate azino-di-3-ethyl-benzthiazolin-sulfonate (Sigma) in 70 mM citrate buffer (pH 4.2) was applied to a new plate and incubated with peptide pins for 10 min at room temperature. The optical density at 415 nm (OD415) and 492 nm was measured in an enzyme-linked immunosorbent assay (ELISA) plate reader (Molecular Device, Sunnyvale, CA). Each assay was repeated more than twice. The cutoff OD415-492 value for positive reaction was the mean OD415-492 + 3 standard deviations of negative control plasma.
RESULTS
Expression of rP44N and rP44N-N, and Western blot analysis. To map the MAb 5C11 epitope, rP44N and rP44N-N were cloned and expressed in E. coli. This expression yielded a 169-amino-acid rP44N (18,550 Da) and a 105-amino-acid rP44N-N (11,635 Da), each of which included 41 amino acids derived from the pET33b(+) vector in the N terminus. The rP44N and rP44N-N were detected as single bands of approximately 19 and 12 kDa, respectively, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Western blot analysis (Fig. 1) showed that both recombinant proteins were recognized by MAb 5C11, indicating that the 5C11 epitope was located within the 64 amino acids encompassing the P44N-N conserved region. MAb 5C11 did not react with E. coli that had been transformed with vector alone.
MAb 5C11 and 3E65 epitopes were exposed to the surface of host cell-free bacteria. In order to determine whether the MAb 5C11 and 3E65 epitopes are exposed to the bacterial surface, host cell-free-organisms were fixed with paraformaldehyde to prevent the penetration of the antibody into the organisms. Both MAbs 5C11 and 3E65 preferentially stained the surface of individual bacteria, resulting in a ring-like labeling pattern (Fig. 2A and B). Normal mouse IgG did not label A. phagocytophilum (Fig. 2D). A rabbit antibody directed against the recombinant A. phagocytophilum NtrX transcription factor (a cytoplasmic protein) did not label prefixed bacteria (Fig. 2C).
When fixed cells were permeabilized by methanol fixation, bacteria were positively labeled with both MAb 5C11 and the anti-NtrX antibody or MAb 3E65 and anti-NtrX (Fig. 2E and F). NtrX (red fluorescence) was in the cytosol and both MAb epitopes (green fluorescence) were on the membrane (Fig. 2E and F). Rabbit preimmune serum did not label organisms (Fig. 2G). These results indicate that both the MAb 5C11 and the MAb 3E65 epitopes are exposed to the bacterial surface. The majority of the A. phagocytophilum population expressed the P44-18 protein. This result was consistent with predominant expression of the p44-18 transcript by the A. phagocytophilum HZ strain in cell culture (34, 38).
Inhibition of binding, development, and infection of A. phagocytophilum with MAbs in vitro. To analyze whether MAb 5C11 and MAb 3E65 block infection of A. phagocytophilum, host cell-free organisms were preincubated with MAbs and then coincubated with HL-60 cells at 37°C for several days to allow the growth of internalized A. phagocytophilum (19). As shown in Fig. 3A, both MAb 5C11 and 3E65 blocked infection, whereas IgG from normal mice, or unrelated mouse MAb isotype IgG2b and IgG1 had no effect on A. phagocytophilum infection. As negative control, host cell-free Ehrlichia chaffeensis, which is the human monocytic ehrlichiosis agent and belongs to the family Anaplasmataceae, was not labeled by either MAb (data not shown), and the infection of HL-60 cells with Ehrlichia chaffeensis was not inhibited by either of the two MAbs (Fig. 3B).
To analyze whether MAb 5C11 and MAb 3E65 block binding of A. phagocytophilum to host cells, host cell-free organisms were preincubated with MAbs and then coincubated with HL-60 cells at room temperature for 15 min with gentle shaking and at 37°C for 45 min to allow binding and internalization of A. phagocytophilum into HL-60 cells. MAb 5C11 almost completely inhibited the binding and internalization of A. phagocytophilum to the host cells compared to normal mouse IgG or a nonrelated mouse MAb (isotype of IgG2b), but MAb 3E65 did not inhibit the A. phagocytophilum binding (Fig. 3C and D). The numbers of extracellular bound bacteria and total host cell-associated bacteria at 1 and 4 h postinoculation were similar with or without MAb 3E65 (Fig. 3E). This result suggested that MAb 3E65 did not inhibit internalization. However, after internalization, transformation from individual bacteria to microcolonies (morulae) was inhibited in HL-60 cells at 16 and 32 h postinoculation (Fig. 3F). The lysosomal fusion was undetectable in any of these bacteria at these time points by double immunofluorescence labeling with anti-CD63 at this time postinoculation (not shown).
Epitope mapping of MAbs 5C11 and 3E65. The epitopes of MAbs 5C11 and 3E65 were determined using peptide-pin ELISA (6). To access the P44 protein hypervariable region that contains the MAb 3E65 epitope, we analyzed the 3D structure of several P44 proteins (P44-18, P44-28, and P44-30) predicted by the Robetta full-length protein prediction program (15, 16). Robetta is a full-chain protein structure prediction server. It parses protein chains into putative domains with the Ginzu protocol, and models those domains either by homology modeling or by ab initio modeling.
The representative predicted structure of the P44 proteins is shown in Fig. 4. By the Robetta program three major domains were predicted in the P44 protein full-length query. (i) Approximately amino acid positions 237 to 437, including the C-terminal half of the central hypervariable region and the C-terminal conserved region, were highly homologous to a -barrel structure of Neisseria meningitidis outer membrane protein NspA (for P44-30 and P44-28, which have PDB BLAST confidence levels of 3.30 and 4.10, respectively, a confidence level of 3.0 predicts almost certainly the right protein folding) or E. coli outer membrane protein A (OmpA) (for P44-18, which has a PDB-BLAST confidence level of 2.27, the confidence level between 2.0 and 3.0 usually predicts the right protein folding). (ii) From the N terminus, an approximately 115- amino-acid sequence, which includes the P44N-N region (from approximately amino acid positions 35 to 98) was characterized by a four-stranded -sheet based on multiple sequence alignment 30 (for P44-18 confidence level was 3.06). (iii) The region from approximately amino acid positions 105 to 240, which includes the region between two absolutely conserved cysteines (C-C region, approximately from amino acid positions 195 to 229) was highly hydrophilic and represents the most variable (nonidentical) domain in the P44s (for P44-18, P44-28, and P44-30, PDB-BLAST confidence levels were 5.14, 16.27, and 16.30, respectively) (21). This C-C region was primarily comprised of turns.
Based on this computer-predicted model of P44 protein structure, overlapping peptides corresponding to the P44-18hv C-C region (30 amino acids) were synthesized for MAb 3E65 epitope analysis. For MAb 5C11 epitope analysis, overlapping peptides corresponding to the P44N-N region (64 amino acids) were synthesized. The MAb 5C11 epitopes were determined to be KFDWNTPD by peptide-pin ELISA analysis (Fig. 5). However, MAb 3E65 bound FAKYIVGA and NRFAKY in the peptide-pin ELISA analysis (Fig. 5). This suggests that the best fit epitope may lie within the core FAKY, with affinity perhaps defined by the flanking regions. We further performed a detailed analysis on NRFAKYIVGA. Comparative analysis using FAKY, FAKYIV, and NRFAKY showed FAKYIV was the strongest epitope peptide recognized. By BLAST search FAKY was detected only in P44-18 but not in any of 86 other P44 proteins predicted to be encoded by A. phagocytophilum HZ (www.tigr.org).
P44N-N and the P44-18hv C-C region epitope mapping of infected horse and mouse plasma. To investigate whether plasma from infected immunocompetent mammals react to these P44 peptides, a peptide-pin ELISA analysis was performed using overlapping peptides derived from the P44N-N region and P44-18hv C-C region. Compared with horse preimmune or normal plasma, all plasma from pooled infected mice, or some plasma from infected horses recognized the MAb 5C11 epitope, and the MAb 3E65 epitope was recognized by all infected mouse and horse plasma (Fig. 6). This suggests that P44-18 was expressed by A. phagocytophilum infecting both host species and that the neutralizing epitope can be recognized in vivo by at least some of infected hosts.
DISCUSSION
The results from this study provide a significant advancement in our understanding of the structure of the P44 protein and neutralizing B-cell epitopes. Park et al. (26) reported dimerization and oligomerization of P44/MSP2 mediated by a disulfide bond. Robetta, using amino acid sequence alone, as with other structure prediction methods, cannot predict influences of other interacting proteins. However, at least three different P44 protein amino acid sequences in our analysis provided the consistent P44 structural model, and the present data were in agreement with the Robetta model indicating that two neutralizing epitopes present within the P44N-N and P44hv C-C regions of naturally folded P44 molecules embedded in the bacterial membrane (thus perhaps in an oligomerized condition) are exposed on the intact bacterial surface and accessible by the MAbs.
Furthermore, MAb 5C11 and 3E65 were found to act as infection neutralizing antibodies in vitro. The result is partially in agreement with a previous work that showed a weak in vitro neutralization with MAbs (26). In the present study, the neutralization mechanisms were found distinct between two MAbs: MAb 5C11 neutralized at the level of binding to HL-60 cells, whereas MAb 3E65 neutralized after internalization of A. phagocytophilum into HL-60 cells. Thus, the P44 hypervariable region may be associated with the critical signals for development of internalized bacteria into a replicating stage to form a morula. Since MAb 3E65 did not block binding, this region does not appear to be involved and may not even be sterically close to the ligand for binding and internalization of A. phagocytophilum into host cells. On the other hand, the epitope of MAb 5C11 appears to be either critical for binding or in close proximity to the ligand of A. phagocytophilum.
Results from the Robetta full-length protein prediction program suggested that the surface-exposed P44 hypervariable C-C region and the P44N-N region are connected to the membrane-embedded -barrel by a short chain of amino acids (positions 236 to 242) that may serve as flexible hinge (Fig. 4A and B). The predicted flexibility of these two exposed regions of the P44 molecule is expected to facilitate intermolecular interaction.
Antibodies directed against the hypervariable region of major surface antigens of bacteria are generally considered to have a strong neutralizing activity; therefore, such antibodies clear this specific antigenic population and allow bacteria with different hypervariable region sequences to become the next dominant population (4). Our previous study showed that the initial P44-18 phenotype is cleared and that new P44 phenotypes sequentially emerged when horses are infected with A. phagocytophilum HZ (34). The present study suggests that the C-C region of P44hv serves as a target for antigenic variation and clearance.
By alignment of multiple P44 proteins, the C-C region is only approximately one-third of the P44 hypervariable region (21). According to the Robetta model, the remaining P44hv region is a part of the -barrel structure and thus predicted not to be exposed to the bacterial surface (Fig. 4). The role of this potentially nonexposed hypervariable region in A. phagocytophilum infection remains to be studied. Our antigenic index analysis, using the Protean program in DNAStar, predicted that the non-surface-exposed region of the P44 hypervariable region is also highly antigenic (21); thus, this region as well as a part of the P44 C-terminal region with predicted high antigenic index may serve as decoy antigens in A. phagocytophilum infection. Similarly, the immunodominant C-terminal domain and one invariable region in the central variable domain (IR6) of Borrelia burgdorferi VlsE are not exposed at the surface of the intact spirochete and thus proposed to serve as decoy epitopes to divert immune responses away from the variable regions (17).
Our study showed that the MAb 5C11 and 3E65 epitopes can be recognized by infected outbred horses and three genetically different mouse strains. It remains to be determined whether these epitopes can be universally recognized by animal and human populations and/or serve as protective immunogens. In addition, several other peptides in the P44N-N region were recognized, and whether these peptides can serve as neutralizing epitopes remains to be analyzed. Furthermore, for more than 80 different P44 hypervariable C-C region amino acid sequences, neutralizing epitopes remain to be verified.
In a previous study, we showed that the p44-18 sequence and genomic locus are conserved among 14 A. phagocytophilum strains from a horse, ticks, and human granulocytic anaplasmosis patients in northeastern states, Wisconsin, and California (20). p44-18 is the major transcript species of A. phagocytophilum HZ that is initially detected in experimentally infected horses and mice, regardless of whether transmission occurs by syringe or by tick attachment (34, 38). Current data further extended these observations, showing specifically recognized peptides within the P44-18hvC-C region by immune plasma.
The structure of the major surface protein 2 (MSP2) of bovine erythrocytic agent Anaplasma marginale is similar to P44, having highly conserved N- and C-terminal regions flanking a central hypervariable region (8). It was reported that B-cell epitopes are present predominantly in the central hypervariable region, and only a few B-cell epitopes are present in the N-terminal conserved region of Msp2 of the A. marginale Florida strain (1). The native MSP2 protein isolated from A. marginale was reported to block hemagglutination of bovine erythrocytes with A. marginale in vitro (23). However, to our knowledge, there have been no reports on the neutralizing B-cell epitopes of MSP2.
In A. marginale, MSP1a and MSP1b are considered ligands for infection of ticks and bovine erythrocytes (7, 9, 23, 24). Passive immunization of cattle with a monoclonal antibody directed against MSP1a neutralizes infection of bovine erythrocytes with A. marginale (25). The serum from rabbits immunized with recombinant MSP1a and MSP1b, either individually or in combination, reduces infection of tick cells with A. marginale (5). However, orthologues of msp1a or msp1b have not been detected in the A. phagocytophilum HZ genome (www.tigr.org). It is possible that in the absence of msp1a and msp1b, p44 (msp2) may have evolved to have a more significant role in the A. phagocytophilum infection of neutrophils.
In summary, our data imply that two separate neutralizing epitopes of P44 proteins are involved in the binding, internalization, and infection of A. phagocytophilum in HL-60 cells, and this may represent the basis for in vivo neutralization with P44N-N and P44hv C-C-specific antibodies. A better understanding of P44 neutralizing epitopes and domains may contribute to the development of new vaccines that can elicit a protective response against A. phagocytophilum.
ACKNOWLEDGMENTS
This research is supported by grant R01AI47407 from the National Institutes of Health.
We appreciate Quan Lin for Robetta analysis and Tzung-Huei Lai for rabbit anti-NtrX antibody.
REFERENCES
1. Abbott, J. R., G. H. Palmer, C. J. Howard, J. C. Hope, and W. C. Brown. 2004. Anaplasma marginale major surface protein 2 CD4+-T-cell epitopes are evenly distributed in conserved and hypervariable regions (HVR), whereas linear B-cell epitopes are predominantly located in the HVR. Infect. Immun. 72:7360-7366.
2. Bakken, J. S., J. S. Dumler, S. M. Chen, M. R. Eckman, L. L. V. Etta, and D. H. Walker. 1994. Human granulocytic ehrlichiosis in the upper Midwest United States. A new species emerging JAMA 272:212-218.
3. Barbet, A. F., P. F. M. Meeus, M. Belanger, M. V. Bowie, J. Yi, A. M. Lundgren, A. R. Alleman, S. J. Wong, F. K. Chu, U. G. Munderloh, and S. D. Jauron. 2003. Expression of multiple outer membrane protein sequence variants from a single genomic locus of Anaplasma phagocytophilum. Infect. Immun. 71:1706-1718.
4. Barbour, A. G. 1990. Antigenic variation of a relapsing fever Borrelia species. Annu. Rev. Microbiol. 44:155-171.
5. Blouin, E. F., J. T. Saliki, J. de la Fuente, J. C. Garcia-Garcia, and K. M. Kocan. 2003. Antibodies to Anaplasma marginale major surface proteins 1a and 1b inhibit infectivity for cultured tick cells. Vet. Parasitol. 111:247-260.
6. Conlan, J. W., I. N. Clarke, and M. E. Ward. 1988. Epitope mapping with solid-phase peptides: identification of type-, subspecies-, species- and genus-reactive antibody binding domains on the major outer membrane protein of Chlamydia trachomatis. Mol. Microbiol. 2:673-679.
7. de la Fuente, J., J. C. Garcia-Garcia, E. F. Blouin, and K. M. Kocan. 2001. Differential adhesion of major surface proteins 1a and 1b of the ehrlichial cattle pathogen Anaplasma marginale to bovine erythrocytes and tick cells. Int. J. Parasitol. 31:145-153.
8. French, D. M., T. F. McElwain, T. C. McGuire, and G. H. Palmer. 1998. Expression of Anaplasma marginale major surface protein 2 variants during persistent cyclic rickettsemia. Infect. Immun. 66:1200-1207.
9. Garcia-Garcia, J. C., J. de la Fuente, G. Bell-Eunice, E. F. Blouin, and K. M. Kocan. 2004. Glycosylation of Anaplasma marginale major surface protein 1a and its putative role in adhesion to tick cells. Infect. Immun. 72:3022-3030.
10. Geysen, H. M. 1990. Molecular technology: peptide epitope mapping and the pin technology. Southeast Asian J. Trop. Med. Public Health 21: 523-533.
11. Ijdo, J. W., C. Wu, S. R. Telford III, and E. Fikrig. 2002. Differential expression of the p44 gene family in the agent of human granulocytic ehrlichiosis. Infect. Immun. 70:5295-5298.
12. Ijdo, J. W., C. Wu, L. A. Magnarelli, and E. Fikrig. 1999. Serodiagnosis of human granulocytic ehrlichiosis by a recombinant HGE-44-based enzyme-linked immunosorbent assay. J. Clin. Microbiol. 37:3540-3544.
13. Kim, H.-Y., J. Mott, N. Zhi, T. Tajima, and Y. Rikihisa. 2002. Cytokine gene expression by peripheral blood leukocytes in horses experimentally infected with Anaplasma phagocytophila. Clin. Diagn. Lab. Immunol. 9:1079-1084.
14. Kim, H. Y., and Y. Rikihisa. 1998. Characterization of monoclonal antibodies to the 44-kilodalton major outer membrane protein of the human granulocytic ehrlichiosis agent. J. Clin. Microbiol. 36:3278-3284.
15. Kortemme, T., and D. Baker. 2004. Computational design of protein-protein interactions. Curr. Opin. Chem. Biol. 8:91-97.
16. Kuhlman, B., and D. Baker. 2004. Exploring folding free energy landscapes using computational protein design. Curr. Opin. Struct. Biol. 14:89-95.
17. Liang, F. T., A. L. Alvarez, Y. Gu, J. M. Nowling, R. Ramamoorthy, and M. T. Philipp. 1999. An immunodominant conserved region within the variable domain of VlsE, the variable surface antigen of Borrelia burgdorferi. J. Immunol. 163:5566-5573.
18. Lin, M., and Y. Rikihisa. 2003. Ehrlichia chaffeensis and Anaplasma phagocytophilum lack genes for lipid A biosynthesis and incorporate cholesterol for their survival. Infect. Immun. 71:5324-5331.
19. Lin, M., and Y. Rikihisa. 2004. Ehrlichia chaffeensis downregulates surface Toll-like receptors 2/4, CD14 and transcription factors PU. 1 and inhibits lipopolysaccharide activation of NF-kappa B, ERK 1/2 and p38 MAPK in host monocytes. Cell Microbiol. 6:175-186.
20. Lin, Q., Y. Rikihisa, R. F. Massung, Z. Woldehiwet, and R. C. Falco. 2004. Polymorphism and transcription at the p44-1/p44-18 genomic locus in Anaplasma phagocytophilum strains from diverse geographic regions. Infect. Immun. 72:5574-5581.
21. Lin, Q., N. Zhi, N. Ohashi, H. W. Horowitz, M. E. Aguero-Rosenfeld, J. Raffalli, G. P. Wormser, and Y. Rikihisa. 2002. Analysis of sequences and loci of p44 homologs expressed by Anaplasma phagocytophila in acutely infected patients. J. Clin. Microbiol. 40:2981-2988.
22. Magnarelli, L. A., J. W. Ijdo, A. E. Van Andel, C. Wu, and E. Fikrig. 2001. Evaluation of a polyvalent enzyme-linked immunosorbent assay incorporating a recombinant p44 antigen for diagnosis of granulocytic ehrlichiosis in dogs and horses. Am. J. Vet. Res. 62:29-32.
23. McGarey, D. J., and D. R. Allred. 1994. Characterization of hemagglutinating components on the Anaplasma marginale initial body surface and identification of possible adhesins. Infect. Immun. 62:4587-4593.
24. McGarey, D. J., A. F. Barbet, G. H. Palmer, T. C. McGuire, and D. R. Allred. 1994. Putative adhesins of Anaplasma marginale: major surface polypeptides 1a and 1b. Infect. Immun. 62:4594-4601.
25. Palmer, G. H., A. F. Barbet, W. C. Davis, and T. C. McGuire. 1986. Immunization with an isolate-common surface protein protects cattle against anaplasmosis. Science 231:1299-1302.
26. Park, J., K. S. Choi, and J. S. Dumler. 2003. Major surface protein 2 of Anaplasma phagocytophilum facilitates adherence to granulocytes. Infect. Immun. 71:4018-4025.
27. Petrovec, M., S. L. Furian, T. A. Zupanc, F. Strle, P. Brouqui, V. Rous, and J. S. Dumler. 1997. Human disease in Europe caused by a granulocytic Ehrlichia species. J. Clin. Microbiol. 35:1556-1559.
28. Rikihisa, Y. 1991. The tribe Ehrlichieae and ehrlichial diseases. Clin. Microbiol. Rev. 4:286-308.
29. Rikihisa, Y., N. Zhi, G. P. Wormser, B. Wen, H. W. Horowitz, and K. E. Hechemy. 1997. Ultrastructural and antigenic characterization of a granulocytic ehrlichiosis agent directly isolated and stably cultivated from a patient in New York State. J. Infect. Dis. 175:210-213.
30. Sadreyev, R. I., and N. V. Grishin. 2004. Estimates of statistical significance for comparison of individual positions in multiple sequence alignments. BMC Bioinformatics 5:106.
31. Reference deleted.
32. Tajima, T., N. Zhi, Q. Lin, Y. Rikihisa, H. W. Horowitz, J. Ralfalli, G. P. Wormser, and K. E. Hechemy. 2000. Comparison of two recombinant major outer membrane proteins of the human granulocytic ehrlichiosis agent for use in an enzyme-linked immunosorbent assay. Clin. Diagn. Lab. Immunol. 7:652-657.
33. van Dobbenburgh, A., A. P. van Dam, and E. Fikrig. 1999. Human granulocutic ehrlichiosis in Western Europe. N. Engl. J. Med. 340:1214-1216.
34. Wang, X., Y. Rikihisa, T.-H. Lai, Y. Kumagai, N. Zhi, and S. M. Reed. 2004. Rapid sequential changeover of expressed p44 genes during the acute phase of Anaplasma phagocytophilum infection in horses. Infect. Immun. 72:6852-6859.
35. Yoshiie, K., H.-Y. Kim, J. Mott, and Y. Rikihisa. 2000. Intracellular infection by the human granulocytic ehrlichiosis agent inhibits human neutrophil apoptosis. Infect. Immun. 68:1125-1133.
36. Zhi, N., N. Ohashi, and Y. Rikihisa. 1999. Multiple p44 genes encoding major outer membrane proteins are expressed in the human granulocytic ehrlichiosis agent. J. Biol. Chem. 274:17828-17836.
37. Zhi, N., N. Ohashi, Y. Rikihisa, H. W. Horowitz, G. P. Wormser, and K. Hechemy. 1998. Cloning and expression of the 44-kilodalton major outer membrane protein gene of the human granulocytic ehrlichiosis agent and application of the recombinant protein to serodiagnosis. J. Clin. Microbiol. 36:1666-1673.
38. Zhi, N., N. Ohashi, T. Tajima, J. Mott, R. W. Stich, D. Grover, S. R. Telford III, Q. Lin, and Y. Rikihisa. 2002. Transcript heterogeneity of the p44 multigene family in a human granulocytic ehrlichiosis agent transmitted by ticks. Infect. Immun. 70:1175-1184.(Xueqi Wang, Takane Kikuch)
ABSTRACT
Anaplasma phagocytophilum is an obligatory intracellular bacterium that causes human granulocytic anaplasmosis. The polymorphic 44-kDa major outer membrane proteins of A. phagocytophilum are dominant antigens recognized by patients and infected animals. However, the ability of anti-P44 antibody to neutralize the infection has been unclear due to a mixture of P44 proteins with diverse hypervariable region amino acid sequences expressed by a given bacterial population and lack of epitope-defined antibodies. Monoclonal antibodies (MAbs) 5C11 and 3E65 are directed to different domains of P44 proteins, the N-terminal conserved region and P44-18 central hypervariable region, respectively. Passive immunization with either MAb 5C11 or 3E65 partially protects mice from infection with A. phagocytophilum. In the present study, we demonstrated that the two monoclonal antibodies recognize bacterial surface-exposed epitopes of naturally folded P44 proteins and mapped these epitopes to specific peptide sequences. The two MAbs almost completely blocked the infection of the A. phagocytophilum population that predominantly expressed P44-18 in HL-60 cells by distinct mechanisms: MAb 5C11 blocked the binding, but MAb 3E65 did not block binding or internalization. Instead, MAb 3E65 inhibited internalized A. phagocytophilum to develop into microcolonies called morulae. Some plasma from experimentally infected horses and mice reacted with these two epitopes. Taken together, these data indicate the presence of at least two distinct bacterial surface-exposed neutralization epitopes in P44 proteins. The results indicate that antibodies directed to certain epitopes of P44 proteins have a critical role in inhibiting A. phagocytophilum infection of host cells.
INTRODUCTION
Human granulocytic anaplasmosis (formerly human granulocytic ehrlichiosis) is an emerging tick-borne zoonosis that has been reported in the United States and Europe (2, 27, 33). Human granulocytic anaplasmosis is caused by infection of an obligatory intracellular bacterium, Anaplasma phagocytophilum, in the family Anaplasmataceae. This bacterium lacks pili, a capsule (28), lipopolysaccharides, and peptidoglycans (18), suggesting that the outer membrane proteins play an important role in its interaction with host granulocytes. A family of proteins with molecular sizes of the 44-kDa range (P44s, also called Msp2s) was shown to be present in the isolated outer membrane fraction of A. phagocytophilum by Western blot analysis and on the surface of A. phagocytophilum within the inclusion by immunogold labeling in the postembedded electron microscopy specimens (14).
P44 proteins are encoded by the p44 (msp2) polymorphic multigene family. Despite its small genome size (1.47 Mb) due to the ongoing reductive genome evolution among members of the family Anaplasmataceae, the A. phagocytophilum genome contains approximately 90 p44 paralogues, suggesting that this large expansion of p44 paralogues has given A. phagocytophilum a survival advantage, perhaps by allowing it to escape host immunoclearance. P44 proteins consist of a single central hypervariable region of approximately 94 amino acid residues, an N-terminal conserved region of approximately 186 amino acids, and a C-terminal conserved region of approximately 146 amino acids; the N- and C-terminal regions flank the central hypervariable region (21, 36). There are three short conserved segments including absolutely conserved two cysteines within the hypervariable region of all predicted P44 proteins (21).
Infected animals develop antibodies directed against the N-terminal conserved region as well as against the hypervariable region (14, 34, 38). P44s undergo antigenic variation during infection in human granulocytic anaplasmosis patients and in experimentally infected horses (3, 34). The hypervariable region of P44 molecules has been assumed to be exposed on the bacterial surface and involved in antigenic variation and immune evasion (3, 14, 21, 34, 36). However, since epitopes of anti-P44 antibodies have never been defined, whether or which part of the hypervariable region or any other regions of naturally folded P44 molecules is exposed to the surface of the intact bacterium has been unknown.
Human granulocytic anaplasmosis patients, unless immunocompromised, generally develop antibodies to P44s; thus, P44s are considered useful antigens for serological diagnosis of human granulocytic anaplasmosis (12, 21, 22, 32, 37). Horses and mice experimentally infected with A. phagocytophilum also develop an antibody to P44s (13, 14, 34). It is less clear whether antibodies to P44s are protective from infection. Ijdo et al. (11) reported lack of protection on day 15 postchallenge in mice immunized with a recombinant P44 protein. Two anti-Msp2 (P44) monoclonal antibodies (MAbs) and a recombinant Msp2 only weakly block A. phagocytophilum binding and infection of HL-60 cells (26). The passive immunization of nave mice with MAbs directed against P44s partially protects mice from infection (14). The results of these studies have given an overall impression that antibodies to directed P44 (Msp2) do not have a significant role in immunoprotection.
However, the previous studies defined neither epitopes of the MAbs or the epitopes of antibodies developed by immunization with the recombinant P44 protein nor p44 species predominantly expressed by the A. phagocytophilum population used to infect the mice or HL-60 cells. Thus, it is unclear whether this poor protection in mice or HL-60 cells is simply due to (i) poor neutralization ability of particular anti-P44 antibodies involved, (ii) lack of surface exposure of the target epitope on the intact bacteria, or (iii) epitope mismatch between anti-P44 antibodies and P44 proteins expressed by the organisms used for infection.
Our MAb 3E65 obtained through screening by immunofluorescence followed by Western blot analysis (14) recognizes a linear epitope within the recombinant hypervariable region of P44-18 protein (33). MAb 5C11 reacts with a linear epitope within the recombinant partial P44-1 protein, which consists of most of the conserved N-terminal region and a part of the hypervariable region of P44-1 (14, 37), with the A. phagocytophilum HZ strain cultured in HL-60 cells at 37°C, which expresses various p44s, but not p44-1 (36), and with diverse P44s derived from several other strains of A. phagocytophilum so far examined (14). Thus, the MAb 5C11 epitope has been considered to be within the conserved P44 N terminus, but not within the hypervariable region of P44-1.
Passive immunization with MAbs 5C11 and 3E65 partially protects nave mice from infection with A. phagocytophilum HZ (14), indicating that P44 proteins contain at least two in vivo neutralizable B-cell epitopes. In the present study, we defined the two neutralization sites on P44 molecules by epitope peptide mapping and used the MAbs to delineate their bacterial surface exposure and inhibitory mechanisms of infection of host cells. The present results support the idea that large expansion of p44 paralogues allows A. phagocytophilum to escape neutralizing antibodies.
MATERIALS AND METHODS
Bacteria. A. phagocytophilum HZ strain isolated from a human granulocytic anaplasmosis patient in 1995 (29) and Ehrlichia chaffeensis Arkansas were cultured in HL-60 cells (a human promyelocytic leukemia cell line) (American Type Culture Collection, Manassas, VA) in RPMI 1640 medium (Gibco-BRL, Grand Island, NY) supplemented with 5% fetal bovine serum (FBS; Bio-technologies Inc., Parker Ford, PA), and 2 mM L-glutamine (Gibco-BRL). Cell cultures were incubated at 37°C in a humidified 5% CO2-95% air atmosphere. The percentage of infected cells was determined by Diff-Quik staining (Baxter Scientific Products, Obetz, OH).
Cloning and expression of rP44N and rP44N-N. Primer pairs TCCGAATTCGACTGGTGGTGCGGGAT and CGCAAGCTTCTAATCAGTCTGCCCTCCGAATTCGACTGGTGGTGCGGGAT and CGCAAGCTTCTACTTAAACCCAATCCGA were designed to clone the DNA fragments encoding recombinant P44N (rP44N; P44N terminal from position 35 to 162, 128 amino acids) and recombinant P44N-N (rP44N-N; N-terminal portion of rP44N, from positions 35 to 98, 64 amino acids) based on the full-length P44-18 sequence at the p44 expression locus (36). The EcoRI and HindIII sites were underlined. PCR products of the expected sizes were obtained and digested with EcoRI and HindIII. These fragments were then ligated into the EcoRI and HindIII sites of the pET33b(+) vector (Novagen Inc., Madison, WI). The resulting plasmid was amplified in Escherichia coli Novablue cells (Novagen). Escherichia coli BL21(DE3) cells (Novagen) were transformed with the recombinant plasmid and induced to express rP44N and rP44N-N with isopropyl-thio--D-galactoside. The recombinant proteins include 41 amino acids at the N terminus derived from the pET33b(+) vector. The rP44N and rP44N-N inclusion bodies were purified using the B-PER II bacterial protein extraction reagent (Pierce, Rockford, IL). The recombinant proteins were then Ni-affinity purified with a His-Select cartridge (Sigma, St. Louis, MO).
Surface labeling of host cell-free A. phagocytophilum with MAb 5C11 and 3E65. Host cell-free organisms were isolated as previously described (35) and cytocentrifuged on glass slides. Organisms were fixed with 2% paraformaldehyde in phosphate-buffered saline (PBS; 137 mM NaCl, 10 mM Na2HPO4, 2.7 mM KCl, 1.8 mM KH2PO4; pH 7.2) for 1 h at room temperature and then incubated with 5C11, 3E65, or normal mouse immunoglobulin G (IgG) only or with both 5C11 and a rabbit antibody directed against the recombinant A. phagocytophilum NtrX transcription factor (T.-H. Lai, Y. Kumagai, and Y. Rikihisa, unpublished data) for 1 h at 37°C. After being washed twice with PBS, cells were incubated with Alexa Fluor 555-conjugated (red fluorescence) goat anti-mouse IgG antibody (Molecular Probes, Eugene, OR) or with both Alexa Fluor 555-conjugated goat anti-rabbit IgG and Alexa Fluor 488-conjugated (green fluorescence) goat anti-mouse IgG antibody (Molecular Probes) for 1 h at 37°C. As the control, host cell-free organisms were fixed with methanol at –20°C for 5 min to permeabilize the organism membrane and then double labeled with both MAb 5C11 and anti-NtrX, with both MAb 3E65 and anti-NtrX, or with both MAb 5C11 and rabbit preimmune serum for 1 h at 37°C. After being washed twice with PBS, cells were incubated with both Alexa Fluor 555-conjugated goat anti-rabbit IgG and Alexa Fluor 488-conjugated goat anti-mouse IgG antibody for 1 h at 37°C. Bacteria were then washed and observed under an Eclipse E400 epifluorescence microscope with a xenon–mercury light source (Nikon, Melville, NY).
Effects of P44 MAbs on binding, internalization, and infection in vitro. For the binding analysis, the host cell-free A. phagocytophilum was freshly prepared from 2.5 x 104 A. phagocytophilum-infected HL-60 cells (>90% cells infected) as previously described (35). The preparation was then incubated with MAbs 5C11 (IgG2b), 3E65 (IgG1) (14), or normal mouse IgG (Sigma)-nonrelated mouse MAbs with the isotype of IgG2b (hybridoma culture supernatant) (final concentration, 1 mg/ml), or with RPMI 1640 medium at room temperature for 30 min with gentle shaking. Each mixture was then added to 2 x 105 uninfected HL-60 cells (final concentration, 1.3 x 106 cells/ml) in RPMI 1640 medium supplemented with 5% FBS and 2 mM L-glutamine. After incubation at room temperature for 15 min with shaking, each mixture was incubated at 37°C in 95% air-5% CO2 for 45 min. Cells were harvested by centrifugation at 750 x g for 5 min and washed with PBS to remove the unbound A. phagocytophilum; this washing procedure was then repeated twice. The mixture was cytocentrifuged and fixed with cold methanol for 5 min and sequentially incubated with horse anti-A. phagocytophilum plasma and Cy3-conjugated anti-horse IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). The number of bound organisms was scored in 100 HL-60 cells in triplicate samples.
For analysis of infection, the host cell-free A. phagocytophilum freshly prepared from each sample of 4 x 105 A. phagocytophilum-infected HL-60 cells (>90% cells infected) as previously described (35), was incubated with various concentration of either MAb 5C11 or 3E65 (14), nonrelated mouse MAb with the isotype of IgG2b, nonrelated mouse MAb with the isotype of IgG1, normal mouse IgG, or RPMI 1640 medium at room temperature for 30 min with gentle shaking. As a negative control, the same number of host cell-free Ehrlichia chaffeensis cells were incubated with MAb 5C11, MAb 3E65, normal mouse IgG or RPMI 1640 medium under the same conditions. Each of the mixtures was added to 2 x 105 uninfected HL-60 cells (final concentration, 106 cells/ml) in RPMI 1640 medium supplemented with 5% FBS and 2 mM L-glutamine and incubated at 37°C in 95% air-5% CO2. After 12 h, the medium was replaced with fresh RPMI 1640 medium supplemented with 5% FBS and 2 mM L-glutamine. The host cell-free organisms were then coincubated with HL-60 cells at 37°C for several days to allow the growth of internalized A. phagocytophilum (19). The infection of A. phagocytophilum and Ehrlichia chaffeensis was evaluated by Diff-Quik staining. The infectivity was scored in 100 HL-60 cells in triplicate samples.
To study the effects of MAbs on the internalization and development of A. phagocytophilum in HL-60 cells, the cell-free organisms were prepared as described above and then incubated with MAb 3E65 (final concentration, 1 mg/ml), or with RPMI 1640 medium at room temperature for 30 min with gentle shaking. Each mixture was then added to uninfected HL-60 cells as described above. After incubation at room temperature for 15 min with shaking, each mixture was incubated at 37°C for 1, 4, 16, and 32 h. At 12 h postinoculation, the medium was replaced with fresh RPMI 1640 medium supplemented with 5% FBS and 2 mM l-glutamine. Cells were harvested and washed as described above, followed by the fixation with 2% paraformaldehyde at room temperature for 1 h. The extracellular bacteria were first stained with horse anti-A. phagocytophilum plasma and Cy3-conjugated goat anti-horse IgG in the absence of saponin, whereas the total extracellular and intracellular bacteria were stained with the same antibodies in the presence of 0.3% saponin.
In order to estimate lysosomal fusion, double immunofluorescence labeling was performed using phycoerythrin-MAb against human CD63 (IgG1; BioLegend, San Diego, CA) and horse anti-A. phagocytophilum plasma (detected with fluorescein isothiocyanate-conjugated goat anti-horse IgG [Jackson ImmunoResearch]) in the presence of 0.3% saponin. All cells were washed three times with PBS to remove unbound antibodies before observation with a Nikon Eclipse E400 fluorescence microscope. Bacteria or inclusions per 100 HL-60 cells were scored in three independent experiments. Statistical analyses were performed by using analysis of variance and the Tukey honestly significant differences test or by Student's t test, and a P of <0.05 was considered significant.
Peptide synthesis and peptide-pin ELISA analysis of MAbs 5C11 and 3E65 and infected horse, mouse, and human plasma. The octamer peptide libraries were synthesized for the P44N-N region (64 amino acids) and the domain bordered by two cysteines in the P44-18 hypervariable (P44-18hv C-C) region (30 amino acids) with three and four overlapping amino acids, respectively, using noncleavable multipin synthesis technology and fluorenylmethoxycarbonyl (Fmoc) chemistry (Mimotopes Pty Ltd., Victoria, Australia) (10). After disruption of the peptide-pins with 0.1 M sodium phosphate buffer containing 1% sodium dodecyl sulfate and 0.1% 2-mercaptoethanol (pH 7.2) and washing with hot water, nonspecific binding sites of pins were blocked with 200 μl of blocking solution containing 2% (wt/vol) bovine serum albumin in PBS-Tween 20 in 96-well plates for 1 h at room temperature.
Sets of peptide-bound pins were washed once with PBS containing 0.1% Tween 20 for 10 min and then incubated with either MAb 5C11 (1:600 dilution), MAb 3E65 (1:100 dilution), or one of the following in blocking solution at 4°C overnight: a 1:50 dilution of horse preimmune plasma from EQ001, EQ005, and EQ006; plasma from seven horses from a region where human granulocytic anaplasmosis is nonendemic (Columbus, Ohio); plasma from horse EQ001 (immunofluorescence titer, 1:320) on day 16 postinoculation of A. phagocytophilum (13); plasma from horse EQ005 (immunofluorescence assay titer, 1:5,000) on day 31 after infected-tick placement; plasma from horse EQ006 (immunofluorescence titer, 1:640) on day 22 postinoculation (33); one of three pooled mouse preimmune plasma samples (for each pool, there were >5 mice); and one of three plasma samples pooled from three infected ICR, C3H/HeN, and C3H/HeJ strain mice (immunofluorescence titer, 1:1,000) on day 31 postinoculation. Immunofluorescence titers of these plasma against A. phagocytophilum were determined as previously described (14).
After being washed four times as described above, horseradish peroxidase-labeled goat anti-mouse or horse IgG (heavy and light chain) (Kirkegaard & Perry Laboratories, Gaithersburg, MD) secondary antibodies were used. All secondary antibodies were diluted 1:500 or 1:200 in 1% (vol/vol) sheep serum (Sigma). The peptide pins were placed in the wells filled with the corresponding secondary antibodies and incubated for 1 h at room temperature. Samples were washed four times, then the horseradish peroxidase substrate azino-di-3-ethyl-benzthiazolin-sulfonate (Sigma) in 70 mM citrate buffer (pH 4.2) was applied to a new plate and incubated with peptide pins for 10 min at room temperature. The optical density at 415 nm (OD415) and 492 nm was measured in an enzyme-linked immunosorbent assay (ELISA) plate reader (Molecular Device, Sunnyvale, CA). Each assay was repeated more than twice. The cutoff OD415-492 value for positive reaction was the mean OD415-492 + 3 standard deviations of negative control plasma.
RESULTS
Expression of rP44N and rP44N-N, and Western blot analysis. To map the MAb 5C11 epitope, rP44N and rP44N-N were cloned and expressed in E. coli. This expression yielded a 169-amino-acid rP44N (18,550 Da) and a 105-amino-acid rP44N-N (11,635 Da), each of which included 41 amino acids derived from the pET33b(+) vector in the N terminus. The rP44N and rP44N-N were detected as single bands of approximately 19 and 12 kDa, respectively, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Western blot analysis (Fig. 1) showed that both recombinant proteins were recognized by MAb 5C11, indicating that the 5C11 epitope was located within the 64 amino acids encompassing the P44N-N conserved region. MAb 5C11 did not react with E. coli that had been transformed with vector alone.
MAb 5C11 and 3E65 epitopes were exposed to the surface of host cell-free bacteria. In order to determine whether the MAb 5C11 and 3E65 epitopes are exposed to the bacterial surface, host cell-free-organisms were fixed with paraformaldehyde to prevent the penetration of the antibody into the organisms. Both MAbs 5C11 and 3E65 preferentially stained the surface of individual bacteria, resulting in a ring-like labeling pattern (Fig. 2A and B). Normal mouse IgG did not label A. phagocytophilum (Fig. 2D). A rabbit antibody directed against the recombinant A. phagocytophilum NtrX transcription factor (a cytoplasmic protein) did not label prefixed bacteria (Fig. 2C).
When fixed cells were permeabilized by methanol fixation, bacteria were positively labeled with both MAb 5C11 and the anti-NtrX antibody or MAb 3E65 and anti-NtrX (Fig. 2E and F). NtrX (red fluorescence) was in the cytosol and both MAb epitopes (green fluorescence) were on the membrane (Fig. 2E and F). Rabbit preimmune serum did not label organisms (Fig. 2G). These results indicate that both the MAb 5C11 and the MAb 3E65 epitopes are exposed to the bacterial surface. The majority of the A. phagocytophilum population expressed the P44-18 protein. This result was consistent with predominant expression of the p44-18 transcript by the A. phagocytophilum HZ strain in cell culture (34, 38).
Inhibition of binding, development, and infection of A. phagocytophilum with MAbs in vitro. To analyze whether MAb 5C11 and MAb 3E65 block infection of A. phagocytophilum, host cell-free organisms were preincubated with MAbs and then coincubated with HL-60 cells at 37°C for several days to allow the growth of internalized A. phagocytophilum (19). As shown in Fig. 3A, both MAb 5C11 and 3E65 blocked infection, whereas IgG from normal mice, or unrelated mouse MAb isotype IgG2b and IgG1 had no effect on A. phagocytophilum infection. As negative control, host cell-free Ehrlichia chaffeensis, which is the human monocytic ehrlichiosis agent and belongs to the family Anaplasmataceae, was not labeled by either MAb (data not shown), and the infection of HL-60 cells with Ehrlichia chaffeensis was not inhibited by either of the two MAbs (Fig. 3B).
To analyze whether MAb 5C11 and MAb 3E65 block binding of A. phagocytophilum to host cells, host cell-free organisms were preincubated with MAbs and then coincubated with HL-60 cells at room temperature for 15 min with gentle shaking and at 37°C for 45 min to allow binding and internalization of A. phagocytophilum into HL-60 cells. MAb 5C11 almost completely inhibited the binding and internalization of A. phagocytophilum to the host cells compared to normal mouse IgG or a nonrelated mouse MAb (isotype of IgG2b), but MAb 3E65 did not inhibit the A. phagocytophilum binding (Fig. 3C and D). The numbers of extracellular bound bacteria and total host cell-associated bacteria at 1 and 4 h postinoculation were similar with or without MAb 3E65 (Fig. 3E). This result suggested that MAb 3E65 did not inhibit internalization. However, after internalization, transformation from individual bacteria to microcolonies (morulae) was inhibited in HL-60 cells at 16 and 32 h postinoculation (Fig. 3F). The lysosomal fusion was undetectable in any of these bacteria at these time points by double immunofluorescence labeling with anti-CD63 at this time postinoculation (not shown).
Epitope mapping of MAbs 5C11 and 3E65. The epitopes of MAbs 5C11 and 3E65 were determined using peptide-pin ELISA (6). To access the P44 protein hypervariable region that contains the MAb 3E65 epitope, we analyzed the 3D structure of several P44 proteins (P44-18, P44-28, and P44-30) predicted by the Robetta full-length protein prediction program (15, 16). Robetta is a full-chain protein structure prediction server. It parses protein chains into putative domains with the Ginzu protocol, and models those domains either by homology modeling or by ab initio modeling.
The representative predicted structure of the P44 proteins is shown in Fig. 4. By the Robetta program three major domains were predicted in the P44 protein full-length query. (i) Approximately amino acid positions 237 to 437, including the C-terminal half of the central hypervariable region and the C-terminal conserved region, were highly homologous to a -barrel structure of Neisseria meningitidis outer membrane protein NspA (for P44-30 and P44-28, which have PDB BLAST confidence levels of 3.30 and 4.10, respectively, a confidence level of 3.0 predicts almost certainly the right protein folding) or E. coli outer membrane protein A (OmpA) (for P44-18, which has a PDB-BLAST confidence level of 2.27, the confidence level between 2.0 and 3.0 usually predicts the right protein folding). (ii) From the N terminus, an approximately 115- amino-acid sequence, which includes the P44N-N region (from approximately amino acid positions 35 to 98) was characterized by a four-stranded -sheet based on multiple sequence alignment 30 (for P44-18 confidence level was 3.06). (iii) The region from approximately amino acid positions 105 to 240, which includes the region between two absolutely conserved cysteines (C-C region, approximately from amino acid positions 195 to 229) was highly hydrophilic and represents the most variable (nonidentical) domain in the P44s (for P44-18, P44-28, and P44-30, PDB-BLAST confidence levels were 5.14, 16.27, and 16.30, respectively) (21). This C-C region was primarily comprised of turns.
Based on this computer-predicted model of P44 protein structure, overlapping peptides corresponding to the P44-18hv C-C region (30 amino acids) were synthesized for MAb 3E65 epitope analysis. For MAb 5C11 epitope analysis, overlapping peptides corresponding to the P44N-N region (64 amino acids) were synthesized. The MAb 5C11 epitopes were determined to be KFDWNTPD by peptide-pin ELISA analysis (Fig. 5). However, MAb 3E65 bound FAKYIVGA and NRFAKY in the peptide-pin ELISA analysis (Fig. 5). This suggests that the best fit epitope may lie within the core FAKY, with affinity perhaps defined by the flanking regions. We further performed a detailed analysis on NRFAKYIVGA. Comparative analysis using FAKY, FAKYIV, and NRFAKY showed FAKYIV was the strongest epitope peptide recognized. By BLAST search FAKY was detected only in P44-18 but not in any of 86 other P44 proteins predicted to be encoded by A. phagocytophilum HZ (www.tigr.org).
P44N-N and the P44-18hv C-C region epitope mapping of infected horse and mouse plasma. To investigate whether plasma from infected immunocompetent mammals react to these P44 peptides, a peptide-pin ELISA analysis was performed using overlapping peptides derived from the P44N-N region and P44-18hv C-C region. Compared with horse preimmune or normal plasma, all plasma from pooled infected mice, or some plasma from infected horses recognized the MAb 5C11 epitope, and the MAb 3E65 epitope was recognized by all infected mouse and horse plasma (Fig. 6). This suggests that P44-18 was expressed by A. phagocytophilum infecting both host species and that the neutralizing epitope can be recognized in vivo by at least some of infected hosts.
DISCUSSION
The results from this study provide a significant advancement in our understanding of the structure of the P44 protein and neutralizing B-cell epitopes. Park et al. (26) reported dimerization and oligomerization of P44/MSP2 mediated by a disulfide bond. Robetta, using amino acid sequence alone, as with other structure prediction methods, cannot predict influences of other interacting proteins. However, at least three different P44 protein amino acid sequences in our analysis provided the consistent P44 structural model, and the present data were in agreement with the Robetta model indicating that two neutralizing epitopes present within the P44N-N and P44hv C-C regions of naturally folded P44 molecules embedded in the bacterial membrane (thus perhaps in an oligomerized condition) are exposed on the intact bacterial surface and accessible by the MAbs.
Furthermore, MAb 5C11 and 3E65 were found to act as infection neutralizing antibodies in vitro. The result is partially in agreement with a previous work that showed a weak in vitro neutralization with MAbs (26). In the present study, the neutralization mechanisms were found distinct between two MAbs: MAb 5C11 neutralized at the level of binding to HL-60 cells, whereas MAb 3E65 neutralized after internalization of A. phagocytophilum into HL-60 cells. Thus, the P44 hypervariable region may be associated with the critical signals for development of internalized bacteria into a replicating stage to form a morula. Since MAb 3E65 did not block binding, this region does not appear to be involved and may not even be sterically close to the ligand for binding and internalization of A. phagocytophilum into host cells. On the other hand, the epitope of MAb 5C11 appears to be either critical for binding or in close proximity to the ligand of A. phagocytophilum.
Results from the Robetta full-length protein prediction program suggested that the surface-exposed P44 hypervariable C-C region and the P44N-N region are connected to the membrane-embedded -barrel by a short chain of amino acids (positions 236 to 242) that may serve as flexible hinge (Fig. 4A and B). The predicted flexibility of these two exposed regions of the P44 molecule is expected to facilitate intermolecular interaction.
Antibodies directed against the hypervariable region of major surface antigens of bacteria are generally considered to have a strong neutralizing activity; therefore, such antibodies clear this specific antigenic population and allow bacteria with different hypervariable region sequences to become the next dominant population (4). Our previous study showed that the initial P44-18 phenotype is cleared and that new P44 phenotypes sequentially emerged when horses are infected with A. phagocytophilum HZ (34). The present study suggests that the C-C region of P44hv serves as a target for antigenic variation and clearance.
By alignment of multiple P44 proteins, the C-C region is only approximately one-third of the P44 hypervariable region (21). According to the Robetta model, the remaining P44hv region is a part of the -barrel structure and thus predicted not to be exposed to the bacterial surface (Fig. 4). The role of this potentially nonexposed hypervariable region in A. phagocytophilum infection remains to be studied. Our antigenic index analysis, using the Protean program in DNAStar, predicted that the non-surface-exposed region of the P44 hypervariable region is also highly antigenic (21); thus, this region as well as a part of the P44 C-terminal region with predicted high antigenic index may serve as decoy antigens in A. phagocytophilum infection. Similarly, the immunodominant C-terminal domain and one invariable region in the central variable domain (IR6) of Borrelia burgdorferi VlsE are not exposed at the surface of the intact spirochete and thus proposed to serve as decoy epitopes to divert immune responses away from the variable regions (17).
Our study showed that the MAb 5C11 and 3E65 epitopes can be recognized by infected outbred horses and three genetically different mouse strains. It remains to be determined whether these epitopes can be universally recognized by animal and human populations and/or serve as protective immunogens. In addition, several other peptides in the P44N-N region were recognized, and whether these peptides can serve as neutralizing epitopes remains to be analyzed. Furthermore, for more than 80 different P44 hypervariable C-C region amino acid sequences, neutralizing epitopes remain to be verified.
In a previous study, we showed that the p44-18 sequence and genomic locus are conserved among 14 A. phagocytophilum strains from a horse, ticks, and human granulocytic anaplasmosis patients in northeastern states, Wisconsin, and California (20). p44-18 is the major transcript species of A. phagocytophilum HZ that is initially detected in experimentally infected horses and mice, regardless of whether transmission occurs by syringe or by tick attachment (34, 38). Current data further extended these observations, showing specifically recognized peptides within the P44-18hvC-C region by immune plasma.
The structure of the major surface protein 2 (MSP2) of bovine erythrocytic agent Anaplasma marginale is similar to P44, having highly conserved N- and C-terminal regions flanking a central hypervariable region (8). It was reported that B-cell epitopes are present predominantly in the central hypervariable region, and only a few B-cell epitopes are present in the N-terminal conserved region of Msp2 of the A. marginale Florida strain (1). The native MSP2 protein isolated from A. marginale was reported to block hemagglutination of bovine erythrocytes with A. marginale in vitro (23). However, to our knowledge, there have been no reports on the neutralizing B-cell epitopes of MSP2.
In A. marginale, MSP1a and MSP1b are considered ligands for infection of ticks and bovine erythrocytes (7, 9, 23, 24). Passive immunization of cattle with a monoclonal antibody directed against MSP1a neutralizes infection of bovine erythrocytes with A. marginale (25). The serum from rabbits immunized with recombinant MSP1a and MSP1b, either individually or in combination, reduces infection of tick cells with A. marginale (5). However, orthologues of msp1a or msp1b have not been detected in the A. phagocytophilum HZ genome (www.tigr.org). It is possible that in the absence of msp1a and msp1b, p44 (msp2) may have evolved to have a more significant role in the A. phagocytophilum infection of neutrophils.
In summary, our data imply that two separate neutralizing epitopes of P44 proteins are involved in the binding, internalization, and infection of A. phagocytophilum in HL-60 cells, and this may represent the basis for in vivo neutralization with P44N-N and P44hv C-C-specific antibodies. A better understanding of P44 neutralizing epitopes and domains may contribute to the development of new vaccines that can elicit a protective response against A. phagocytophilum.
ACKNOWLEDGMENTS
This research is supported by grant R01AI47407 from the National Institutes of Health.
We appreciate Quan Lin for Robetta analysis and Tzung-Huei Lai for rabbit anti-NtrX antibody.
REFERENCES
1. Abbott, J. R., G. H. Palmer, C. J. Howard, J. C. Hope, and W. C. Brown. 2004. Anaplasma marginale major surface protein 2 CD4+-T-cell epitopes are evenly distributed in conserved and hypervariable regions (HVR), whereas linear B-cell epitopes are predominantly located in the HVR. Infect. Immun. 72:7360-7366.
2. Bakken, J. S., J. S. Dumler, S. M. Chen, M. R. Eckman, L. L. V. Etta, and D. H. Walker. 1994. Human granulocytic ehrlichiosis in the upper Midwest United States. A new species emerging JAMA 272:212-218.
3. Barbet, A. F., P. F. M. Meeus, M. Belanger, M. V. Bowie, J. Yi, A. M. Lundgren, A. R. Alleman, S. J. Wong, F. K. Chu, U. G. Munderloh, and S. D. Jauron. 2003. Expression of multiple outer membrane protein sequence variants from a single genomic locus of Anaplasma phagocytophilum. Infect. Immun. 71:1706-1718.
4. Barbour, A. G. 1990. Antigenic variation of a relapsing fever Borrelia species. Annu. Rev. Microbiol. 44:155-171.
5. Blouin, E. F., J. T. Saliki, J. de la Fuente, J. C. Garcia-Garcia, and K. M. Kocan. 2003. Antibodies to Anaplasma marginale major surface proteins 1a and 1b inhibit infectivity for cultured tick cells. Vet. Parasitol. 111:247-260.
6. Conlan, J. W., I. N. Clarke, and M. E. Ward. 1988. Epitope mapping with solid-phase peptides: identification of type-, subspecies-, species- and genus-reactive antibody binding domains on the major outer membrane protein of Chlamydia trachomatis. Mol. Microbiol. 2:673-679.
7. de la Fuente, J., J. C. Garcia-Garcia, E. F. Blouin, and K. M. Kocan. 2001. Differential adhesion of major surface proteins 1a and 1b of the ehrlichial cattle pathogen Anaplasma marginale to bovine erythrocytes and tick cells. Int. J. Parasitol. 31:145-153.
8. French, D. M., T. F. McElwain, T. C. McGuire, and G. H. Palmer. 1998. Expression of Anaplasma marginale major surface protein 2 variants during persistent cyclic rickettsemia. Infect. Immun. 66:1200-1207.
9. Garcia-Garcia, J. C., J. de la Fuente, G. Bell-Eunice, E. F. Blouin, and K. M. Kocan. 2004. Glycosylation of Anaplasma marginale major surface protein 1a and its putative role in adhesion to tick cells. Infect. Immun. 72:3022-3030.
10. Geysen, H. M. 1990. Molecular technology: peptide epitope mapping and the pin technology. Southeast Asian J. Trop. Med. Public Health 21: 523-533.
11. Ijdo, J. W., C. Wu, S. R. Telford III, and E. Fikrig. 2002. Differential expression of the p44 gene family in the agent of human granulocytic ehrlichiosis. Infect. Immun. 70:5295-5298.
12. Ijdo, J. W., C. Wu, L. A. Magnarelli, and E. Fikrig. 1999. Serodiagnosis of human granulocytic ehrlichiosis by a recombinant HGE-44-based enzyme-linked immunosorbent assay. J. Clin. Microbiol. 37:3540-3544.
13. Kim, H.-Y., J. Mott, N. Zhi, T. Tajima, and Y. Rikihisa. 2002. Cytokine gene expression by peripheral blood leukocytes in horses experimentally infected with Anaplasma phagocytophila. Clin. Diagn. Lab. Immunol. 9:1079-1084.
14. Kim, H. Y., and Y. Rikihisa. 1998. Characterization of monoclonal antibodies to the 44-kilodalton major outer membrane protein of the human granulocytic ehrlichiosis agent. J. Clin. Microbiol. 36:3278-3284.
15. Kortemme, T., and D. Baker. 2004. Computational design of protein-protein interactions. Curr. Opin. Chem. Biol. 8:91-97.
16. Kuhlman, B., and D. Baker. 2004. Exploring folding free energy landscapes using computational protein design. Curr. Opin. Struct. Biol. 14:89-95.
17. Liang, F. T., A. L. Alvarez, Y. Gu, J. M. Nowling, R. Ramamoorthy, and M. T. Philipp. 1999. An immunodominant conserved region within the variable domain of VlsE, the variable surface antigen of Borrelia burgdorferi. J. Immunol. 163:5566-5573.
18. Lin, M., and Y. Rikihisa. 2003. Ehrlichia chaffeensis and Anaplasma phagocytophilum lack genes for lipid A biosynthesis and incorporate cholesterol for their survival. Infect. Immun. 71:5324-5331.
19. Lin, M., and Y. Rikihisa. 2004. Ehrlichia chaffeensis downregulates surface Toll-like receptors 2/4, CD14 and transcription factors PU. 1 and inhibits lipopolysaccharide activation of NF-kappa B, ERK 1/2 and p38 MAPK in host monocytes. Cell Microbiol. 6:175-186.
20. Lin, Q., Y. Rikihisa, R. F. Massung, Z. Woldehiwet, and R. C. Falco. 2004. Polymorphism and transcription at the p44-1/p44-18 genomic locus in Anaplasma phagocytophilum strains from diverse geographic regions. Infect. Immun. 72:5574-5581.
21. Lin, Q., N. Zhi, N. Ohashi, H. W. Horowitz, M. E. Aguero-Rosenfeld, J. Raffalli, G. P. Wormser, and Y. Rikihisa. 2002. Analysis of sequences and loci of p44 homologs expressed by Anaplasma phagocytophila in acutely infected patients. J. Clin. Microbiol. 40:2981-2988.
22. Magnarelli, L. A., J. W. Ijdo, A. E. Van Andel, C. Wu, and E. Fikrig. 2001. Evaluation of a polyvalent enzyme-linked immunosorbent assay incorporating a recombinant p44 antigen for diagnosis of granulocytic ehrlichiosis in dogs and horses. Am. J. Vet. Res. 62:29-32.
23. McGarey, D. J., and D. R. Allred. 1994. Characterization of hemagglutinating components on the Anaplasma marginale initial body surface and identification of possible adhesins. Infect. Immun. 62:4587-4593.
24. McGarey, D. J., A. F. Barbet, G. H. Palmer, T. C. McGuire, and D. R. Allred. 1994. Putative adhesins of Anaplasma marginale: major surface polypeptides 1a and 1b. Infect. Immun. 62:4594-4601.
25. Palmer, G. H., A. F. Barbet, W. C. Davis, and T. C. McGuire. 1986. Immunization with an isolate-common surface protein protects cattle against anaplasmosis. Science 231:1299-1302.
26. Park, J., K. S. Choi, and J. S. Dumler. 2003. Major surface protein 2 of Anaplasma phagocytophilum facilitates adherence to granulocytes. Infect. Immun. 71:4018-4025.
27. Petrovec, M., S. L. Furian, T. A. Zupanc, F. Strle, P. Brouqui, V. Rous, and J. S. Dumler. 1997. Human disease in Europe caused by a granulocytic Ehrlichia species. J. Clin. Microbiol. 35:1556-1559.
28. Rikihisa, Y. 1991. The tribe Ehrlichieae and ehrlichial diseases. Clin. Microbiol. Rev. 4:286-308.
29. Rikihisa, Y., N. Zhi, G. P. Wormser, B. Wen, H. W. Horowitz, and K. E. Hechemy. 1997. Ultrastructural and antigenic characterization of a granulocytic ehrlichiosis agent directly isolated and stably cultivated from a patient in New York State. J. Infect. Dis. 175:210-213.
30. Sadreyev, R. I., and N. V. Grishin. 2004. Estimates of statistical significance for comparison of individual positions in multiple sequence alignments. BMC Bioinformatics 5:106.
31. Reference deleted.
32. Tajima, T., N. Zhi, Q. Lin, Y. Rikihisa, H. W. Horowitz, J. Ralfalli, G. P. Wormser, and K. E. Hechemy. 2000. Comparison of two recombinant major outer membrane proteins of the human granulocytic ehrlichiosis agent for use in an enzyme-linked immunosorbent assay. Clin. Diagn. Lab. Immunol. 7:652-657.
33. van Dobbenburgh, A., A. P. van Dam, and E. Fikrig. 1999. Human granulocutic ehrlichiosis in Western Europe. N. Engl. J. Med. 340:1214-1216.
34. Wang, X., Y. Rikihisa, T.-H. Lai, Y. Kumagai, N. Zhi, and S. M. Reed. 2004. Rapid sequential changeover of expressed p44 genes during the acute phase of Anaplasma phagocytophilum infection in horses. Infect. Immun. 72:6852-6859.
35. Yoshiie, K., H.-Y. Kim, J. Mott, and Y. Rikihisa. 2000. Intracellular infection by the human granulocytic ehrlichiosis agent inhibits human neutrophil apoptosis. Infect. Immun. 68:1125-1133.
36. Zhi, N., N. Ohashi, and Y. Rikihisa. 1999. Multiple p44 genes encoding major outer membrane proteins are expressed in the human granulocytic ehrlichiosis agent. J. Biol. Chem. 274:17828-17836.
37. Zhi, N., N. Ohashi, Y. Rikihisa, H. W. Horowitz, G. P. Wormser, and K. Hechemy. 1998. Cloning and expression of the 44-kilodalton major outer membrane protein gene of the human granulocytic ehrlichiosis agent and application of the recombinant protein to serodiagnosis. J. Clin. Microbiol. 36:1666-1673.
38. Zhi, N., N. Ohashi, T. Tajima, J. Mott, R. W. Stich, D. Grover, S. R. Telford III, Q. Lin, and Y. Rikihisa. 2002. Transcript heterogeneity of the p44 multigene family in a human granulocytic ehrlichiosis agent transmitted by ticks. Infect. Immun. 70:1175-1184.(Xueqi Wang, Takane Kikuch)