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Infection and Immunity, January 2005, p. 79-87, Vol. 73, No. 1
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.1.79-87.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Ehrlichia chaffeensis Expresses Macrophage- and Tick Cell-Specific 28-Kilodalton Outer Membrane Proteins

Vijayakrishna Singu, Haijie Liu, Chuanmin Cheng, and Roman R. Ganta^*

Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, Kansas

Received 26 July 2004/ Returned for modification 26 August 2004/ Accepted 9 September 2004

Top Abstract Introduction Materials and Methods Results Discussion References

 
Ehrlichia chaffeensis, a tick-transmitted rickettsial agent, causes human monocyte/macrophage-tropic ehrlichiosis. In this study, proteomic approaches were used to demonstrate host cell-specific antigenic expression by E. chaffeensis. The differentially expressed antigens include those from the 28-kDa outer membrane protein (p28-Omp) multigene locus. The proteins expressed in infected macrophages are the products of p28-Omp19 and p28-Omp20 genes, whereas in tick cells, the protein expressed is the p28-Omp14 gene product. The differentially expressed proteins are posttranslationally modified by phosphorylation and glycosylation to generate multiple expressed forms. Host cell-specific protein expression is not influenced by growth temperatures and is reversible. Host cell-specific protein expression coupled with posttranslational modifications may be a hallmark for the pathogen's adaptation to a dual-host life cycle and its persistence.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Tick-borne infections are responsible for many emerging pathogenic bacterial diseases that significantly impact the health of humans and other vertebrates (13, 40). These include spirochete infections, such as those caused by the Lyme disease pathogen Borrelia burgdorferi, and rickettsial infections by the human monocytic ehrlichiosis agent, Ehrlichia chaffeensis, the human granulocytic anaplasmosis agent, Anaplasma phagocytophilum, and the bovine anaplasmosis agent, Anaplasma marginale (5, 9, 12, 13, 27). Little is known about the morphological adaptation of these bacteria in dual hosts. Understanding the molecular events of bacterial adaptation to tick and vertebrate hosts will be valuable for the development of effective control strategies. Recent studies on B. burgdorferi suggest that the pathogen's adaptation involves the expression of unique host-specific surface antigens (12, 15, 44). Recent studies on Anaplasma species also suggest the differential expression of surface proteins in tick and mammalian hosts (22, 25, 43). To date, no studies have described how Ehrlichia species adapt to arthropod and vertebrate host environments.

E. chaffeensis is an emerging infectious agent that causes severe and potentially fatal disease in immune-compromised and elderly people (37, 38). This organism has a multigene locus spanning 22 tandemly arranged genes that encode immunodominant 28-kDa outer membrane proteins (p28-Omp) (7, 26, 34, 34, 35, 41, 42, 48). The protein coding sequences of the p28-Omp genes have four long stretches of conserved regions separated by three highly variable, hydrophilic regions where the dominant immunogenic B-cell epitopes are located (42). Multigene families homologous to the p28-Omp multigene locus of E. chaffeensis have also been reported for other, closely related Ehrlichia species; such as Ehrlichia canis and Ehrlichia ruminantium (references in reference 7). The protein-coding sequences of the p28-Omp genes of Ehrlichia species show significant homology to the multigene families of the outer membrane proteins MSP2 and p44 of Anaplasma species (24, 42). Recent reports suggest that the MSP2 and p44 gene loci of Anaplasma spp. are involved in generating antigenic variation during infection (2, 4, 39). Expression of unique variants from these multigene families have also been reported for Anaplasma spp. in their tick and vertebrate hosts (25, 43). The structural features of the p28-Omp loci of E. chaffeensis are also similar to variant surface antigen genes of the pathogenic bacteria B. burgdorferi and Neisseria gonorrhoeae (21, 45, 50).

It is of significant interest to determine if the proteins from the p28 multigene loci are differentially expressed in tick and vertebrate hosts to aid in the adaptation and/or persistence of Ehrlichia species, including E. chaffeensis. Several recent studies that evaluated the transcripts of the p28-Omp locus from E. chaffeensis both in vitro and in vivo by nonquantitative reverse transcription-PCR (RT-PCR) methods revealed conflicting results (7, 26, 47). Long et al. (26) and Unver et al. (47) suggested that at least 16 paralogs from this locus are transcriptionally active. For some of the genes compared, the transcripts detected by Unver et al. differed from those reported by Long et al. In a recent study, Cheng et al. also performed extensive RT-PCR analysis to examine expression from the p28-Omp multigene loci of several E. chaffeensis isolates and presented evidence that it is difficult to draw meaningful conclusions regarding gene expression and the contributions of the multigene locus to the pathogen's adaptation or persistence from RNA analysis, as judged by RT-PCR methods (7).

In the present study, we utilized proteomic approaches to identify expressed proteins and to resolve the controversy that existed with RT-PCR-based RNA analysis. We present data in support of the novel macrophage- and tick cell-specific differential protein expression from the p28 multigene locus and other regions of the genome of E. chaffeensis. In this report, we also present evidence for the existence of extensive posttranslational modifications in the differentially expressed antigens from the p28-Omp locus.

Top Abstract Introduction Materials and Methods Results Discussion References

 
In vitro cultivation of E. chaffeensis. The E. chaffeensis Arkansas isolate was continuously cultivated in the canine macrophage cell line DH82 at 37 or 34°C (6). E. chaffeensis in DH82 cells was also cultivated at 25°C. This was accomplished by transferring a 50%-infected flask grown at 37°C to a 25°C incubator and maintaining it for 96 h. The organism was also cultivated in a tick cell line, ISE6, at 34°C by following a protocol similar to that described in reference 31.

When infectivity reached 80 to 90%, 15 ml of culture from a 75-cm² (T-75) confluent flask was harvested and centrifuged at 15,560 x g for 15 min. The resulting culture pellet was resuspended in 10 ml of SPK buffer (0.5 M K₂HPO₄, 0.5 M KH₂PO₄, and 0.38 M sucrose) and sonicated twice at a setting of 6.5 for 30 s in a Sonic Dismembrator (Fisher Scientific, Pittsburgh, Pa.) to release E. chaffeensis. The lysates were centrifuged at 100 x g for 5 min, and the supernatants were filtered sequentially through 5- and 3-µm-pore-size sterile membrane filter assemblies (Millipore, Billerica, Mass.). E. chaffeensis organisms from the filtrates were concentrated by centrifuging for 15 min at 15,560 x g, and the pellets were washed twice with SPK buffer.

Protein preparation for 2DE. Total-protein preparations from purified E. chaffeensis, uninfected DH82 cells, and uninfected ISE6 cells were made for use in the two-dimensional gel electrophoresis (2DE) analysis by following the protocol described in reference 20. Briefly, purified organisms were lysed in the presence of 0.5 ml of lysis buffer (9 M urea, 2% Triton X-100, 3.24 mM dithiothreitol [DTT], 8 mM phenylmethylsulfonyl fluoride, and 0.2% ampholyte [pH 3 to 10] [Amersham Pharmacia Biotech, Piscataway, N.J.]) for 1 h at room temperature and then precipitated by using 2 volumes of an ice-cold acetone-trichloroacetic acid mixture (4:1). The final protein pellet was dissolved in 100 µl of the sample buffer {9 M urea, 0.2% ampholyte [pH 3 to 10], 2% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate [CHAPS], 65 mM DTT, 0.52% Triton X-100, and 0.001% bromophenol blue}. Protein concentrations were determined by using a reducing-agent-compatible and detergent-compatible protein assay kit (Bio-Rad, Hercules, Calif.).

2DE and Western blot analysis. One hundred micrograms of total protein was resolved at 20°C in the first dimension by isoelectric focusing (IEF) in a Multiphor II electrophoresis system (Amersham Pharmacia Biotech) using 11-cm-long, pH 3 to 10, precast immobilized pH gradient strips (Amersham Biosciences, Uppsala, Sweden). The IEF parameters were 300 V, 2 mA, and 5 W for 1 min, followed by 3,500 V, 2 mA, and 5 W for 245 min. At the end of the IEF, the strips were equilibrated sequentially for 15 min each in 1 ml of equilibration buffer I (375 mM Tris-HCl [pH 8.8], 6 M urea, 2% sodium dodecyl sulfate [SDS], 2% DTT) and buffer II (375 mM Tris-HCl [pH 8.8], 6 M urea, 2% SDS, 2.72 mg of iodoacetamide/ml, 0.001% bromophenol blue). Subsequently, second-dimension SDS-polyacrylamide gel electrophoresis analysis was performed on the strips in a Criterion cell apparatus by using 4 to 20% polyacrylamide gradient gels (Bio-Rad) for 2 h at 74 V at room temperature in a 50 mM Tris-glycine buffer. The 2DE resolved gels were stained by using a silver-staining kit (Bio-Rad). The 2DE gels were also used to perform Western blot analysis with E. chaffeensis polyclonal sera. Polyclonal sera were obtained from wild-type C57BL6 mice 21 to 24 days after infection with either macrophage-grown or tick cell-grown E. chaffeensis by following protocols described previously (18).

Silver-stained gels were digitalized by using an HP Scanjet 7400c scanner (Hewlett-Packard, Houston, Tex.), and the images were analyzed by using Phoretix 2DE image analysis software (Phoretix 2D Evolution [version 2003.01] package; Nonlinear Dynamics, Durham, N.C.) to perform comparisons of 2D gels so as to correct the gel running artifacts and to detect spots in the gels that were aligned. The software determines qualitative and quantitative differences in the expressed proteins between different experimental groups. It examines and selects all spots showing apparent changes in expression. They are subjected to analysis of variance, a statistical method of analysis used to assess differences between different samples.

LC-MS/MS. Proteins excised from the 2DE gels were digested with trypsin and used for liquid chromatography-electrospray ionization-ion trap mass spectrometry (LC-MS/MS) analysis. This analysis was performed at the Stanford University mass spectrometry facility (http://mass-spec.stanford.edu/), and the peptide fragment fingerprint data were subjected to an NCBI-nr database search by using the Mascot MS/MS ion search programs (www.matrixscience.com).

Phosphoprotein or glycoprotein staining and image analysis. Phosphoprotein and glycoprotein staining was performed for proteins resolved by 2DE by using the Pro-Q Diamond phosphoprotein and Pro-Q Emerald 300 staining methods, respectively, according to the manufacturer's protocol (Molecular Probes, Eugene, Oreg.). Images of the stained gels were captured by using a Typhoon multimode imager 9410 (Amersham Biosciences Corp., Piscataway, N.J.) or a UV transilluminator (Spectroline, Westburg, N.Y.) and a Kodak (Rochester, N.Y.) EDAS 290 system. The gels were restained with SYPRO Ruby according to the manufacturer's protocol (Molecular Probes) to detect total protein present in the gels. Candy Cane glycoprotein and Peppermint Stick phosphoprotein molecular weight standards from Molecular Probes were used for protein size determinations.

Enzymatic deglycosylation and dephosphorylation. Two hundred micrograms of E. chaffeensis proteins in the lysis buffer was digested with 5 U of PNGase F [peptide-N-glycosidase F, peptide-N⁴ (N-acetyl-ß-glucosaminyl) asparagine amidase F] (Sigma Chemical Co., St. Louis, Mo.) at 37°C for 72 h. PNGase F hydrolyzes the N-linked glycan moieties from asparagine in a protein. At the end of incubation, the reaction was stopped by boiling for 5 min and the deglycosylated proteins were precipitated by adding 2 volumes of an ice-cold acetone-trichloroacetic acid mixture (4:1). The dephosphorylation protocol was essentially the same as the deglycosylation protocol except that protein phosphatase (PPase) (New England Biolabs, Beverly, Mass.) was used in place of PNGase F and the incubation time was reduced to 30 min. PPase releases phosphate groups from serine, threonine, or tyrosine residues from a protein. To remove glycan and phosphate moieties, the protein fractions were digested sequentially with PNGase F and PPase. The dephosphorylated and deglycosylated proteins were resolved and analyzed by 2DE gel analysis and immunoblotting as described above.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Proteome analysis identifies unique host-specific protein expression of E. chaffeensis. E. chaffeensis organisms grown in macrophages and tick cells were purified so that they were free of host cells and cellular proteins. The proteomes of three independently purified samples of macrophage- and tick cell-derived E. chaffeensis were assessed by 2DE analyses. The reproducibility of the data for each host cell-specific E. chaffeensis proteome and the absence of contaminating host cell proteins were confirmed by analyzing the 2D gels with the aid of image analysis software (Fig. 1). Comparison of the proteomes of E. chaffeensis from macrophages to those from tick cells revealed considerable visible differences, in addition to detection of several overlapping protein spots in the gels (Fig. 2). The obvious differences include the presence of many resolved proteins migrating between pH 7 and 9 in the macrophage-derived E. chaffeensis that are absent in the tick cell-derived proteome. Similarly, the tick cell-derived proteome contained significantly more proteins migrating between pH 6 and 7. The proteins in the resolved gels for macrophage- and tick cell-derived E. chaffeensis were estimated and compared by use of an image analysis program. The total numbers of proteins for macrophage- and tick cell-derived E. chaffeensis were very similar, ranging from 363 to 449 and 382 to 428, respectively. The degree of variation in the proteins detected in different samples within each host-specific E. chaffeensis proteome was 23 to 26%. In agreement with the differences detectable in resolved gels, image analysis also revealed 70 to 71% differences between the proteins of E. chaffeensis organisms grown in macrophages and those grown in tick cells.

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FIG. 1. Comparison of the 2DE gel-resolved proteomes of host cells and E. chaffeensis from infected host cells. Three independent experiments were performed, and the data for one experimental comparison of the macrophage proteome and E. chaffeensis from infected macrophages, together with the warped image generated by using Phoretix 2D image analysis software, are shown in panels A to C. Similarly, the tick cell proteome was compared with that of E. chaffeensis grown in tick cells. Approximate locations of the pH values of the protein migration and the protein molecular weight standards are presented above and to the right of the gels, respectively. The E. chaffeensis proteomes differed significantly from those of host cells. Three independently isolated samples of E. chaffeensis proteomes resolved on 2DE gels were also analyzed by using image analysis software. Data for comparison of two samples and the warped image generated by using Phoretix 2D are shown in panels D to F. Similarly, the tick cell-grown E. chaffeensis proteome was analyzed.

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FIG. 2. E. chaffeensis proteomes are host cell specific. Scanned images of silver-stained 2D gels of E. chaffeensis proteomes are shown. Approximate locations of the pI values of the protein migration and the protein molecular weight standards are presented above and to the right of the gels, respectively. The experiment was performed on three independently isolated samples, and the reproducibility of the data was confirmed by analysis with Phoretix 2D image analysis software. Total numbers of proteins detected by the software in each gel for E. chaffeensis grown in macrophages and tick cells ranged from 363 to 449 and 382 to 428, respectively. The 2D software analysis identified as much as 50% host-cell-specific differences in the proteins expressed by E. chaffeensis. (Insets) Western blot data for the proteins spanning 28 to 30 kDa, identified by using E. chaffeensis polyclonal sera.

Western blot analysis identifies host-specific, unique immunoreactive proteins of E. chaffeensis. Examination of the macrophage-derived E. chaffeensis proteome by Western blot analysis using plasma raised against E. chaffeensis grown in macrophages identified several immunoreactive proteins (data not shown). They included two rows of five 28-kDa antigens (three major and two minor protein spots) and six 30-kDa antigens (three major and three minor protein spots) which migrated between pH 4.5 and 5.5 (Fig. 2A, inset). Similar analysis of tick cell-grown E. chaffeensis by using the macrophage-derived E. chaffeensis plasma also detected several immunoreactive proteins. The immunodominant proteins in tick cell-grown E. chaffeensis contained only one row of five 30-kDa antigens that were resolved in the same pH region (Fig. 2B, inset).

Mass spectrometry analysis identifies unique host-specific protein expression from the p28-Omp locus. We reasoned that the multiple immunoreactive proteins spanning the 28- to 30-kDa region may represent those expressed from the p28-Omp multigene locus. To test this hypothesis, we investigated the identities of these proteins by LC-MS/MS analysis. Analysis of the immunoreactive proteins spanning the 28- to 30-kDa region identified 6 out of 11 macrophage-derived antigens as products of the p28-Omp19 gene (Fig. 3). One of these proteins also showed identity to the p28-Omp20 gene product (Fig. 3). The analysis was repeated two additional times using proteins isolated from different batches of E. chaffeensis. The conclusions remained the same, except that in one of the experiments, the p28-Omp20 protein was not detected. Similar triplicate analysis of the five 30-kDa immunoreactive proteins of tick cell-derived E. chaffeensis revealed the proteins to be the product of the p28-Omp14 gene (Fig. 4).

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FIG. 3. LC-MS/MS analysis of macrophage-grown E. chaffeensis immunodominant proteins. Immunodominant proteins with molecular sizes ranging from 28 to 30 kDa were cored from the 2DE gels and used for generating LC-MS/MS data. The analysis was performed on three independent samples; panel A gives results from one sample. (A) List of identified proteins along with the number of matched peptides for each protein and their total sequence coverage. (B) Protein spots that showed identity by LC-MS/MS analysis. (C) Mascot data search results for one protein band (B1) that showed identity to p28-Omp19 with 38% sequence coverage generated from the matched peptides (boldfaced). (D) Diagram representing the genes of the p28-Omp locus. The nomenclature of the genes from this locus is presented as discussed in reference 7. Genes for the protein products identified are highlighted with black arrowhead boxes.

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FIG. 4. LC-MS/MS analysis of tick cell-grown E. chaffeensis immunodominant proteins. Immunodominant 30-kDa proteins were cored from the 2DE gels for LC-MS/MS analysis. The analysis was performed on three independent samples. (A) Results from one sample, including the proteins identified, with the number of matched peptides and percent coverage. (B) Protein spots that showed identity by LC-MS/MS analysis. (C) Mascot data search results for one protein band having identity to p28-Omp14 with 48% sequence coverage (boldfaced). (D) The p28-Omp locus, presented as described in the legend to Fig. 3. Genes for the protein products identified are highlighted with black arrowhead boxes.

Multiple p28-Omp proteins observed on 2DE proteome blots resulted from posttranslational modifications. The appearance of multiple proteins spanning the 28- to 30-kDa region but showing identity to p28-Omp19 and -20 proteins for macrophage-derived E. chaffeensis and to p28-Omp14 protein for tick cell-derived E. chaffeensis required further analysis. We tested the hypothesis that posttranslational modifications generate proteins with variable molecular weights and pIs from an expressed p28-Omp antigen. Glycoprotein and phosphoprotein staining revealed several host cell-specific glycoproteins and phosphoproteins in the proteomes of E. chaffeensis (Fig. 5). They included proteins migrating in the region spanning the p28-Omp antigens (Fig. 5).

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FIG. 5. Glycoprotein and phosphoprotein staining. E. chaffeensis proteomes from macrophages and tick cells were resolved by 2DE, and gels were stained with the total-protein, glycoprotein, or phosphoprotein stain. Proteins spanning the 28- to 30-kDa region are boxed. Protein standards are shown to the right of the gels.

To present more-direct evidence for glycosylation and phosphorylation in the p28-Omp antigens, we conducted experiments to test the hypothesis that deglycosylation reduces the molecular mass of the 30-kDa proteins to 28 kDa and that dephosphorylation changes the pH of the proteins, making them less acidic. Specifically, if the E. chaffeensis proteomes were deglycosylated, we expected to see the disappearance of the upper row of immunoreactive p28-Omp proteins from macrophage-derived E. chaffeensis, while deglcosylation coupled with dephosphorylation would reduce all of the p28 protein bands to one spot with the lower molecular mass and the higher pH. The results from these experiments are presented in Fig. 6. Enzymatic deglycosylation followed by 2DE and Western blot analysis of macrophage-grown E. chaffeensis detected only one row of 28-kDa proteins (Fig. 6B). Enzymatic deglycosylation also reduced the tick cell-derived E. chaffeensis 30-kDa immunoreactive proteins by about 2 kDa (Fig. 6E). Enzymatic removal of both glycan and phosphate moieties resulted in the disappearance of nearly all the 28- to 30-kDa proteins, except for one that had a higher pH and a lower molecular size for both macrophage- and tick-derived E. chaffeensis (Fig. 6C and F). These lone protein spots were determined by LC-MS/MS analysis to be the products of the p28-Omp19 and -20 genes for macrophage-grown E. chaffeensis and of the p28-Omp14 gene for tick cell-grown E. chaffeensis.

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FIG. 6. Enzymatic deglycosylation and dephosphorylation. Proteins spanning 28 to 30 kDa from a 2DE gel of E. chaffeensis proteomes analyzed by Western blotting before or after deglycosylation, alone or in combination with dephosphorylation, are presented. (A to C) E. chaffeensis proteins from organisms grown in a macrophage cell line; (D to F) proteins from tick cell cultures. Western blot data before enzymatic treatment (A and D), after enzymatic deglycosylation (B and E), and after both deglycosylation and dephosphorylation (C and F) are presented. LC-MS/MS analysis of the lone protein spots detected in panels C and F showed identities to the p28-Omp19 and -20 and p28-Omp14 gene products, respectively.

Host cell-specific protein expression can be reverted. If the expressed antigens are truly host cell specific, one would expect the reversal of E. chaffeensis protein expression when the pathogen grown in the tick cell is used to infect the macrophage, or vice versa. This was investigated by transferring tick cell-derived E. chaffeensis into the macrophage cell line for regrowth and examining the cultured organisms by 2DE and Western blot analysis (Fig. 7). Examination by LC-MS/MS analysis showed that the reverted organisms had a proteome map similar to that originally observed for macrophage-grown E. chaffeensis, including expression of p28-Omp19.

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FIG. 7. Host-specific protein expression by E. chaffeensis is reversible. Shown are the region-spanning proteins of 28 to 30 kDa from Western blots of a 2DE gel of E. chaffeensis grown in tick cell culture and transferred to DH82 culture. The reverted organisms had a proteome similar to that originally observed in the macrophage. They include the immunodominant p28-Omp antigens. LC-MS/MS analysis identified the excised protein spots from reverted culture as the product of p28-Omp19.

Differential host-specific protein expression is independent of the temperature at which E. chaffeensis is grown. Since the invertebrate host is poikilothermic, the temperature at which E. chaffeensis is grown in a tick is expected to fluctuate. In this study, we also investigated whether the observed proteome differences between macrophage- and tick cell-derived E. chaffeensis organisms can be generated by simple alteration of the growth temperature. Proteome maps of E. chaffeensis cultured at three different temperatures—25, 34, and 37°C—in macrophages remained essentially the same, with minor differences in the expressed proteins (data not shown). Immunoblot analysis of the proteomes revealed a temperature-dependent variation in nonglycosylated proteins among the p28-Omp antigens (Fig. 8). At 34°C, the majority of the proteins are in a glycosylated form. At 25°C, only glycosylated proteins were detected. The expressed p28-Omp antigens of E. chaffeensis grown at 34 and 25°C were identified as the gene products of p28-Omp19, a finding similar to that for growth at 37°C (determined by LC-MS/MS analysis). Similarly, tick cell-grown E. chaffeensis at 34 and 37°C showed little proteome variation (data not shown). p28-Omp14 expression by E. chaffeensis also remained constant in the tick cell at these two different growth temperatures (determined by LC-MS/MS analysis).

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FIG. 8. Effect of growth temperature on p28-Omp protein expression. Western blots of the proteome region spanning proteins from 28 to 30 kDa from a 2DE gel of DH82 cell-derived E. chaffeensis grown at three different temperatures are presented. LC-MS/MS analysis revealed that host-specific p28-Omp protein expression remained constant in E. chaffeensis at three different growth temperatures (p28-Omp19), except that the expressed p28-Omp proteins are more efficiently glycosylated at lower temperatures.

Top Abstract Introduction Materials and Methods Results Discussion References

 
In this study, we utilized proteomic approaches to demonstrate novel host cell-specific protein expression of several E. chaffensis antigens, including those from the p28-Omp multigene locus. Multiple 2DE analysis revealed that E. chaffeensis organisms cultivated in a macrophage differed considerably from those grown in a tick cell. E. chaffeensis grown in macrophages expressed numerous proteins that were resolved in the alkaline pH range of 7 to 9, whereas in the tick cell the organism expressed considerably more proteins that were resolved in the slightly acidic pH range of 5 to 7. Although very few visible differences were found in 2D gels of different preparations of E. chaffeensis from the same infected host cell, the output data from the 2DE analysis software revealed about 25% variation among different protein preparations. Taking this sample-to-sample variation into account, approximately 50% of E. chaffeensis proteins were differentially expressed in the two host cells. These data are supported by the noticeable differences in 2D gels of host cell-specific proteomes. We do not expect to see any host cell proteins in the E. chaffeensis proteomes, because the purification method involves several steps to eliminate host cell contamination. Nonetheless, a more accurate estimate of true differences in the expressed proteins of E. chaffeensis relative to host cells, including the total number of proteins, can be made only following a detailed global mass spectrometry analysis.

Western blot analysis of the 2DE proteome gels further supported the existence of host cell-specific differences. The majority of the immunoreactive proteins in macrophage-derived E. chaffeensis migrated in two rows between 28 and 30 kDa, with pH ranging from 4.5 to 5.5. The immunodominant proteins of tick cell-grown E. chaffeensis resolved as one row in the 30-kDa region within the same pH range. Mass spectrometry analysis established the identities of these immunodominant proteins as the products of the p28-Omp19 and -20 genes for macrophage-derived E. chaffeensis and of the p28-Omp14 gene for tick cell-derived E. chaffeensis. Expression of p28-Omp14 protein by E. chaffeensis in tick cells is in agreement with the detection of the transcript for this protein in E. chaffeensis-infected Amblyomma americanum ticks (47).

In a recent study, it was reported that the murine host immunized with macrophage-derived E. chaffeensis contained p28-Omp19 antibodies, indirect evidence suggesting that the p28-Omp19 protein is expressed by the organism in infected macrophages. Our present mass spectrometry data for macrophage-derived E. chaffeensis confirm the previous conclusion about the expression of the p28-Omp19 gene product. In the previous study, it was also suggested that the macrophage-derived E. chaffeensis sera do not cross-react with antigens other than the p28-Omp19 products of the multigene locus, such as the recombinant antigen of p28-Omp gene 16 (7). However, in the present study, we present evidence that sera raised against E. chaffeensis from infected macrophages also reacted with the p28-Omp14 gene product in tick cell-derived E. chaffeensis. This is contradictory to the previous hypothesis. Recognition of the p28-Omp14 antigen by sera raised against macrophage-derived E. chaffeensis may have resulted from the presence of cross-reactive antibodies to this antigen; however, we may have failed to identify other expressed antigens from the p28-Omp locus that may lack the cross-reactive epitopes in the tick cell-derived E. chaffeensis. To rule out this possibility, we reevaluated the 2D gel blots of the tick cell-derived proteome by using murine sera raised against the organisms grown in tick cells. The immunodominant proteins recognized in the 28-kDa region by using the tick cell-derived E. chaffeensis sera remained the same as those observed with the E. chaffeensis sera from infected macrophages, except that the second row of proteins was also detected (data not shown). These data confirm that there is only one p28-Omp expressed antigen, p29-Omp14, present in E. chaffeensis in infected tick cells.

The pIs estimated from the predicted amino acid sequences of all 22 genes of the p28-Omp multigene locus range from pH 4.55 to 9.76. The predicted pIs for the expressed proteins from the p28-Omp14, -19, and -20 genes are 4.76, 5.11, and 5.17, respectively. The immunodominant p28-Omp proteins detected in E. chaffeensis from infected macrophage and tick cells, identified in the present study, migrated between pIs 4.5 and 5.5. These values are well within the expected pI values for these proteins.

Recent reports on evaluation of the transcriptional activity of the genes from the p28-Omp multigene locus, as judged by nonquantitative RT-PCR methods, revealed conflicting results and suggested that as many as 16 genes are expressed (7, 26, 47). Based on the peptide enzyme-linked immunosorbent assay analysis, Zhang et al. suggested that all 22 proteins from the p28-Omp multigene locus are expressed concurrently during persistent E. chaffeensis infection in the canine host (51). Despite the detection of many transcriptionally active genes by RT-PCR and the presence of antibody titers for all 22 proteins, N-terminal amino acid sequence analysis, reported earlier by two independent groups, supports the expression of primarily one gene product from the p28-Omp locus for the pathogen grown in a macrophage cell line (26, 35). The p28-Omp19 gene product detected in our present study by LC-MS/MS analysis in macrophage-derived E. chaffeensis is in complete agreement with these N-terminal amino acid sequence data.

The lack of translated products independent of the presence of transcripts in E. chaffeensis is similar to the reports in the literature for a related rickettsia, A. marginale (2, 25). An operon of A. marginale contains an expressed MSP2 gene and three upstream open reading frames, OpAG1, OpAG2, and OpAG3 (2). Analysis of RNA from this operon suggests the presence of a polycistronic transcript that contains all four open reading frames (2). However, the translated products are differentially expressed in vertebrate and tick hosts (25). Particularly, the OpAG1 protein is not expressed in either tick or vertebrate hosts, while OpAG3 is expressed only in the vertebrate host, not in the tick host. The presence of only one or two proteins reported in the present study for the p28-Omp genes in E. chaffeensis, independent of the detection of multiple transcripts reported in the literature, may reflect the fact that not all transcripts can translate into proteins. Most importantly, our study and those of A. marginale clearly demonstrate that assessment of gene activity in rickettsiales requires analysis at the protein level.

It is well documented in the literature that tick-transmitted rickettsiales, including E. chaffeensis, establish persistent infections (1, 8, 10, 11, 14, 17, 46, 47, 49, 52). However, little is known about how these bacteria persist in their dual hosts. Host cell-specific differential protein expression in vertebrate and tick cells, including those from the p28-Omp multigene locus encoding outer membrane antigens, may be one of the mechanisms that aid the persistence of E. chaffeensis in tick and vertebrate hosts. Differential gene expression by B. burgdorferi, a tick-transmitted spirochete, within a host has been demonstrated and appears to contribute to its adaptation in dual hosts (12, 15, 44). B. burgdorferi expresses outer surface protein A (OspA) when it enters its tick host, Ixodes scapularis, and continues to produce abundant OspA within the resting tick (references in reference 12). During its transmission from tick to mammals and while it is in the vertebrate host, expression is switched from OspA to OspC. E. chaffeensis may employ similar strategies of differential host-specific protein expression from the p28-Omp locus in support of its persistent infection and dual-host life cycle in vertebrate and tick hosts. The p28-Omp antigens of E. chaffeensis share considerable homology with the MSP2 outer membrane proteins of A. marginale (42). The MSP2 proteins are also encoded by a multigene locus, and recent reports suggest that antigenic variation in MSP2 expression contributes to the persistence of A. marginale in its vertebrate host (2, 16). The possible role of the p28-Omp antigens in generating antigenic variants in E. chaffeensis, similar to those in A. marginale, can now be critically evaluated in vivo by using the proteomic methods described here.

In this study, evidence is also presented that several E. chaffeensis proteins are posttranslationally modified by phosphorylation and glycosylation. This is the first report to present evidence for phosphorylation. Glycosylation has been reported for another outer membrane protein of E. chaffeensis, the p120 antigen, and for the major surface protein 1 of A. marginale (19, 28). Glycoproteins in prokaryotes may have many specific functions, including maintenance of the cell shape, protein stability, protection against proteolysis, and adherence to host cells (19, 28, 29). Phosphorylation of proteins can impact a wide variety of cellular activities, ranging from events such as the activation of membrane proteins to transport signals to alter a protein's function (23, 36). Little is known about how either glycosylation or phosphorylation contributes to E. chaffeensis growth in vertebrate and tick hosts. The evidence presented here about glycosylation supports previous reports of its existence in rickettsiales. The significance of glycosylation and phosphorylation for E. chaffeensis remains to be studied. The generation of multiple posttranslational forms derived from one expressed protein is also a novel finding and opens up the possibility that these forms may differ in their function and antigenicity.

Because in mammals the body temperature remains constant while a tick's body temperature is mostly influenced by its surroundings, it can be argued that differences in E. chaffeensis protein expression result from temperature variations. In this study, we observed only minor global changes in the E. chaffeensis proteome in response to varying temperatures. Temperature has no effect on the p28-Omp expressed antigens. One obvious difference noted is that lower temperatures facilitated glycosylation efficiently. This is in contrast to reports on temperature-dependent differential expression of B. burgdorferi outer surface proteins (33). Differential host-specific protein expression independent of the growth temperature has also been reported for A. marginale and A. phagocytophilum outer membrane proteins (22).

The tick cell line ISE6, used in this study, is derived from embryonic cells of I. scapularis (32). Although I. scapularis is not the transmitting vector of E. chaffeensis, our study demonstrates that the ISE6 cell line serves as a good starting point for examining the impact of the tick cell environment on antigenic expression by E. chaffeensis. ISE6 is one of the two tick cell lines that have been extensively used for initial assessment of the mechanisms involved in differential expression of antigens by rickettsial agents (3, 22, 30-32).

In summary, this is the first study to report macrophage- and tick cell-specific antigenic expression and the generation of multiple protein forms by posttranslational modifications for a tick-borne rickettsial pathogen. Host-specific protein expression by E. chaffeensis, together with the generation of multiple posttranslational forms, may be a hallmark for this organism's adaptation and persistence in dual hosts. The proteomic methods employed in this study will be valuable for translational research with E. chaffeensis to further examine the host-specific differences in its natural vertebrate and tick hosts.

 
We thank Ulrike Munderloh, University of Minnesota, St. Paul, for providing the ISE6 cell line and E. chaffeensis-infected ISE6 cells. We also acknowledge the technical assistance of Andrew Guzzetta (Stanford University Mass Spectrometry Facility, Stanford, Calif.) in mass spectrometry analysis.

This study was supported by National Institutes of Health grants RR17686, AI50785, and AI55052.

 
* Corresponding author. Mailing address: Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, 1800 Denison Ave., Manhattan, KS 66506. Phone: (785) 532-4612. Fax: (785) 532-4851. E-mail: rganta{at}vet.k-state.edu.

Kansas Agricultural Experiment Station contribution 05-9-J.

Editor: J. T. Barbieri

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Infection and Immunity, January 2005, p. 79-87, Vol. 73, No. 1
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