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Infection and Immunity, November 2008, p. 4823-4832, Vol. 76, No. 11
0019-9567/08/$08.00+0     doi:10.1128/IAI.00484-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Total, Membrane, and Immunogenic Proteomes of Macrophage- and Tick Cell-Derived Ehrlichia chaffeensis Evaluated by Liquid Chromatography-Tandem Mass Spectrometry and MALDI-TOF Methods{triangledown} ,{dagger}

Gwi-Moon Seo,1 Chuanmin Cheng,1 John Tomich,2 and Roman R. Ganta1*

Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine,1 Department of Biochemistry, College of Arts and Sciences, Kansas State University, Manhattan, Kansas 665062

Received 18 April 2008/ Returned for modification 5 June 2008/ Accepted 7 August 2008


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ABSTRACT
 
Ehrlichia chaffeensis, a tick-transmitted rickettsial, is the causative agent of human monocytic ehrlichiosis. To examine protein expression patterns, we analyzed total, membrane, and immunogenic proteomes of E. chaffeensis originating from macrophage and tick cell cultures. Total proteins resolved by one-dimensional gel electrophoresis and subjected to liquid chromatography-electrospray ionization ion trap mass spectrometry allowed identification of 134 and 116 proteins from macrophage- and tick cell-derived E. chaffeensis, respectively. Because a majority of immunogenic proteins remained in the membrane fraction, individually picked total and immunogenic membrane proteins were also surveyed by liquid chromatography-tandem mass spectrometry and matrix-assisted laser desorption ionization-time of flight methods. The analysis aided the identification of 48 additional proteins. In all, 278 genes of the E. chaffeensis genome were verified as functional genes. They included genes for DNA and protein metabolism, energy metabolism and transport, membrane proteins, hypothetical proteins, and many novel proteins of unknown function. The data reported in this study suggest that the membrane of E. chaffeensis is very complex, having many expressed proteins. This study represents the first and the most comprehensive analysis of E. chaffeensis-expressed proteins. This also is the first study confirming the expression of nearly one-fourth of all predicted genes of the E. chaffeensis genome, validating that they are functionally active genes, and demonstrating that classic shotgun proteomic approaches are feasible for tick-transmitted intraphagosomal bacteria. The identity of novel expressed proteins reported in this study, including the large selection of membrane and immunogenic proteins, will be valuable in elucidating pathogenic mechanisms and developing effective prevention and control methods.


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INTRODUCTION
 
Several tick-borne rickettsial diseases having significant impact on the health of people and classified under emerging diseases have been reported in recent years (11). These diseases include human monocytic ehrlichiosis and human ewingii ehrlichiosis, caused by Ehrlichia chaffeensis and Ehrlichia ewingii, respectively, and human granulocytic anaplasmosis caused by Anaplasma phagocytophilum (2, 3, 8, 11).

Tick-transmitted rickettsiales including Ehrlichia species may have evolved strategies of dual host adaptation to aid their continued survival in both vertebrate and invertebrate host environments. Differential protein expression may be an important adaptation mechanism used by arthropod-borne pathogens to support growth and persistence in invertebrate and vertebrate host cell environments (13). Differential expression is reported for tick-transmitted bacterial pathogens, such as Anaplasma marginale, E. chaffeensis, Ehrlichia canis, and Borrelia burgdorferi (9, 10, 12, 29, 34, 36, 37). Differential expression in B. burgdorferi has also been shown to aid adaptation of its transition between arthropod vector and mammalian host (10, 12, 34).

A multigene locus containing 22 tandemly arranged genes that encode for immunodominant 28-kDa outer membrane proteins, commonly referred to as p28-Omps, has been described for E. chaffeensis and other closely related Ehrlichia species (15, 26, 28, 32, 33). Recently, we presented evidence for macrophage- and tick cell-specific differential expression of proteins in E. chaffeensis that included expression from a subset of proteins representing the p28-Omp locus (36, 37). Our protein analysis data for a subset of E. chaffeensis proteins differed from previous studies that assessed gene expression by reverse transcription-PCR (RT-PCR). RNA analysis suggested the presence of multiple transcripts made from different genes spanning the p28-Omp locus of E. chaffeensis in macrophage-derived pathogen (5, 21, 39). The discrepancy can be better addressed with a comprehensive proteome analysis.

Recent developments in proteome research greatly aid understanding of the complexity of numerous expressed proteins of an organism (23). The E. chaffeensis genome is about 1,176 kb, and the annotated whole-genome analysis predicted 1,115 open reading frames (ORFs) as likely genes present in the genome (17). However, it is not clear how many of the predicted ORFs are truly expressed and how many may be differentially expressed by the bacteria in vertebrate and tick host environments. We hypothesize that E. chaffeensis expresses only a subset of proteins at a given stage of its life cycle. Recently, we estimated E. chaffeensis expressed proteins to be about 360 to 450 (36) and reported that the majority of expressed proteins appear to represent a group differentially expressed in response to vertebrate and tick cell environments, some having multiple posttranslated forms (36, 37).

In the present study, we used proteomic approaches to establish the identity of numerous expressed proteins of E. chaffeensis originating from macrophage and tick cell environments. The comprehensive analysis aided identification of 278 E. chaffeensis expressed proteins, including numerous membrane, immunogenic, and secretory proteins. Analysis also identified 55 proteins made from hypothetical protein genes and several proteins with unknown function. The identity of novel expressed proteins reported in the present study will be valuable in elucidating pathogenic mechanisms and developing effective methods of prevention and control.


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MATERIALS AND METHODS
 
In vitro cultivation of E. chaffeensis in macrophage and tick cell lines. E. chaffeensis Arkansas isolate was cultivated at 37°C in the canine macrophage cell line, DH82, as described previously (4). This isolate was also cultivated in two tick cell lines, ISE6 and AAE2, but at 34°C in the absence of CO2 as described by Munderloh et al. (25). The ISE6 and AAE2 cell lines are derived from Ixodes scapularis and Amblyomma americanum embryos, respectively (25, 36, 37).

E. chaffeensis total protein preparation and one-dimensional gel electrophoresis (1-DE) analysis. Harvesting of E. chaffeensis cultures, purification, and protein preparations for total protein analysis were described earlier (37).

Preparation of cytoplasmic and membrane fractions of E. chaffeensis proteins and Western blot analysis. Membrane and cytoplasmic protein fractions were prepared from purified E. chaffeensis as described by Molloy et al. (24). Purified E. chaffeensis was resuspended in 10 ml of 50 mM Tris-HCl (pH 7.5) containing 7 mM phenylmethylsulfonyl fluoride and 0.07 mg of DNase I. Cells were sonicated twice at a setting of 12 for 30 s by using a Sonic Dismembrator (Fisher Scientific, Pittsburgh, PA). Lysate was centrifuged twice at 2,500 x g for 10 min to remove unbroken cells, and the supernatant was transferred to a clean tube, diluted with freshly prepared 0.1 M sodium carbonate buffer (pH 11.0) to a final volume of 6 ml, and stirred slowly on ice for 1 h. Carbonate-treated membrane fractions were collected by ultracentrifugation at 115,500 x g for 1 h at 4°C in a Beckman MLS-50 rotor (Beckman Coulter, Fullerton, CA). Membrane fractions were resuspended in 50 mM Tris-HCl buffer (pH 7.5), and the ultracentrifugation step was repeated for 20 min at 4°C. The supernatant was transferred to a clean tube, and the membrane pellet fraction was solubilized immediately in membrane buffer (7 M urea, 2 M thiourea, 0.5% Triton X-100, 40 mM Tris-HCl [pH 7.5], and 30 mM dithiothreitol). Protein concentrations were determined with an RCDC kit according to the manufacturer's instructions (Bio-Rad, Hercules, CA).

About 100 µg of total, membrane, and cytoplasmic proteins was resolved in an 18.3-by-20-cm-long, manually prepared 10 to 20% sodium dodecyl sulfate-polyacrylamide gel by subjecting it to 250 V for 5 h at room temperature using 50 mM Tris-glycine buffer. Resolved proteins from the gels were either visualized after staining with Coomassie brilliant blue G-250 for use in mass spectrometry (MS) analysis or transferred to a nitrocellulose membrane for use in Western blot analysis (Amersham Biosciences, Piscataway, NJ). Electroblot transfer was performed by using a Trans-Blot cell as described by the manufacturer (Bio-Rad). Western blot analysis was performed (36) with E. chaffeensis polyclonal sera obtained from C57BL/6J mice at 21 to 24 days after experimental infection with DH82 macrophage- or ISE6 tick cell-propagated E. chaffeensis (13).

Liquid chromatography-electrospray ionization ion trap MS (GeLC-MS/MS) analysis of 1-DE-resolved E. chaffeensis proteins. E. chaffeensis total proteins resolved on a 1-DE gels were sliced into several subfractions; proteins present in each fraction of the gel were identified by performing in-gel trypsin digestion, followed by nanocapillary liquid chromatography-tandem MS (LC-MS/MS) analysis and electrosprayed into a quadrupole time-of-flight (TOF) tandem mass spectrometer. In-gel digestion and LC-MS/MS analysis were performed at the Stanford University Mass Spectrometry Facility as we described earlier (36, 37). Peptide fragment fingerprint data generated from the MS/MS analysis were subjected to a database search against E. chaffeensis genome data using the MASCOT MS/MS ion search programs. Peptide masses having identity with E. chaffeensis proteins were further analyzed by searching against NCBI-nr database. The parameters used to assign positive protein identification included up to one missed cleavage, fixed modifications of propionamide (Cys), a peptide mass tolerance of ±2-Da fragment, an MS/MS tolerance of ±0.8 Da, and a peptide charge of 2+ or 3+. The percentage of protein coverage was based on peptide hits with a MASCOT ion score cutoff of 20.

Matrix-assisted laser desorption ionization (MALDI)-TOF MS analysis of E. chaffeensis membrane proteins. Membrane proteins from Coomassie blue-stained gels were picked individually by using PROTEINEER spII with spControl 3.0 software (Bruker Daltonics, Bremen, Germany) according to the manufacturer's protocol. Coomassie blue-stained proteins were digested as described by Shevchenko et al. (35). An aliquot of in-gel-digested solution was mixed with an equal volume of a saturated solution of {alpha}-cyano-4-hydroxycinnamic acid in 50% aqueous acetonitrile, and 1 µl of mixture was spotted onto a target plate. Protein analysis was performed with a Bruker UltraFlex II MALDI-TOF using Prespotted AnchorChip with 384 matrix spots. MALDI-TOF spectra were externally calibrated using a combination of nine standard peptides: bradykinin 1-7 (757.39 Da), angiotensin II (1,046.54 Da), angiotensin I (1,296.68 Da), neurotensin (1,672.91 Da), renin substrate (1,758.93 Da), ACTH clip 1-17 (2,093.08 Da), ACTH clip 18-39 (2,465.19 Da), ACTH clip 1-24 (2,932.58 Da), and ACTH clip 7-38 (3,657.92 Da), spotted onto positions adjacent to the samples. Protein identification was carried out by manual and automatic comparison of experimentally generated monoisotopic values of peptides using MASCOT with a tolerance of 0.2 to 0.5 Da and one missed cleavage, and oxidation of methionine was allowed.

Subcellular localization prediction and gene ontology analysis. To obtain the subcellular localization and biological processes of identified proteins, we used a bioinformatics program, BII, developed by Wang et al. (40) (http://protein.bii.a-star.edu.sg/localization/gram-negative/). Gene ontology for the E. chaffeensis genome is used as reported by Hotopp et al. (17).


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RESULTS
 
Total protein analysis by GeLC-MS/MS identifies many common and uniquely expressed proteins of E. chaffeensis originating from macrophage and tick cells. The Arkansas isolate of the pathogen cultivated in a macrophage cell line, DH82, and in an I. scapularis tick cell line, ISE6, were resolved on by 1-DE (Fig. 1). Resolved proteins were divided into several subfractions and subjected to in-gel trypsin digestion. The resulting peptide mixtures were analyzed by GeLC-MS/MS. A database search of output data identified 134 and 116 proteins from macrophage- and tick cell culture-derived E. chaffeensis, respectively. (A list of all identified proteins with the GenBank numbers is presented in Table S1 in the supplemental material.)


Figure 1
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  		FIG. 1. 1-DE-resolved proteins of E. chaffeensis. The molecular sizes of the protein standards are shown on the left (lane L). Numbered sections 1 to 6 on the right side of each of the resolved protein lanes refer to sliced fractions of the resolved proteins used for performing the GeLC-MS/MS analysis.

Identified proteins included many common cell functional proteins, such as metabolic pathways, energy production, transport, DNA and protein synthesis, and protein fate, and many outer membrane- and cell envelope-associated proteins. Large numbers of proteins from both macrophage- and tick cell-derived E. chaffeensis are also encoded from hypothetical protein genes. Proteins expressed in macrophage- or tick cell-derived bacteria belong to categories such as cell envelope/outer membrane proteins, hypothetical proteins, proteins with unknown function, and cofactor and vitamin biosynthesis. Analysis identified only two paralogs of the p28-Omp multigene locus (p28-Omp1 and p28-Omp14) in tick cell-derived E. chaffeensis, whereas peptides spanning 18 of the 22 paralogs (all but p28-Omp1', p28-Omp4, p28-Omp9, and p28-Omp21) were identified in macrophage-derived E. chaffeensis. Matched peptides were located within variable regions of the p28-Omps (Table 1 and see also Fig. S1 in the supplemental material). The p28-Omp paralogs in macrophage-derived E. chaffeensis also included the p28-Omp14 gene product. The 120-kDa glycosylated outer membrane protein was detected only in tick cell-derived E. chaffeensis (31). Both macrophage- and tick cell-derived E. chaffeensis proteins included the type IV secretion system (TFSS)-mediated ankyrin repeat protein homologue of A. phagocytophilum (18, 20, 30).


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  		TABLE 1. Matched peptide locations of the p28-Omp genes identified by MS

Membrane protein analysis. The outer membrane is likely the primary contact between the pathogen and host cell; thus, membrane proteins are likely targets for host response and vaccine development (1, 22, 28). To identify additional membrane-expressed proteins, E. chaffeensis total protein extracts were fractionated into membrane and cytoplasmic subfractions and resolved on 1-DE gels, and proteins were evaluated by immunoblot analysis to determine the location of immunogenic proteins (data for an ISE6 tick cell-derived E. chaffeensis are presented in Fig. 2). A majority of the immunogenic proteins were detected in the membrane fractions (Fig. 2). Thus, membrane fractions made from E. chaffeensis originating from macrophage culture, vector (A. americanum) and nonvector (I. scapularis) tick cells (AAE2 and ISE6, respectively), were resolved on a 1-DE gel and evaluated by Western blotting (Fig. 3). Macrophage-derived E. chaffeensis proteins differed considerably from those isolated from tick cells. E. chaffeensis proteins originating from both vector and nonvector tick cell lines had a very similar 1-DE-resolved membrane and immunogenic protein pattern (Fig. 3A and B, lanes 2 and 3). Few apparent differences were noted among immunogenic E. chaffeensis proteins from vector and nonvector tick cells. These include the expression of a ~100-kDa protein having higher expression level in AAE2 and the detection of an ~35-kDa protein in AAE2 culture-derived E. chaffeensis that is absent in ISE6 culture-grown bacteria (Fig. 3).


Figure 2
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  		FIG. 2. 1-DE-resolved total, cytoplasmic, and membrane fractions of ISE6 tick cell culture-derived E. chaffeensis proteins. Lanes: L, protein standards; T, total proteins; M, membrane fraction; C, cytoplasmic fraction. The molecular sizes of the protein standards were shown on the left. (A) Coomassie blue G-250-stained gel. (B) Western blot data for the proteins resolved as in panel A.


Figure 3
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  		FIG. 3. 1-DE-resolved membrane fractions of E. chaffeensis proteins. Lanes: L, protein standards (molecular sizes are shown on the left); 1, macrophage (DH82) culture-derived membrane fraction; 2, AAE2 tick cell culture-derived membrane fraction; 3, ISE6 tick cell culture-derived membrane fraction. (A) Coomassie blue G-250-stained gel. (B) Western blot data for the proteins resolved as in panel A. Numbers with arrowhead lines refer to protein spots excised for performing MALDI-TOF analysis.

All visible macrophage-derived membrane proteins from Coomassie blue-stained gels were picked individually and subjected to MALDI-TOF analysis (Table 2). Since the protein expression pattern is very similar, a subset of immunogenic membrane proteins from vector and nonvector tick cell-derived E. chaffeensis was initially assessed by MALDI-TOF. Analysis revealed identical proteins expressed in E. chaffeensis grown in both types of tick cells (Fig. 3B). Subsequently, all visible spots from Coomassie blue-stained gels of either AAE2 or ISE6 tick cell-derived E. chaffeensis membrane proteins were assessed to maximize membrane protein identification (Table 2). Membrane protein analysis aided in the identification of many novel proteins, some of which were also identified during total proteome analysis.


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  		TABLE 2. Membrane proteins of E. chaffeensis analyzed by MALDI-TOF

Immunoblots were also aligned with Coomassie blue-stained gels and, independent of the presence or absence of visibly detectable protein spots, regions corresponding to Western blot positives were excised from gels and subjected to MALDI-TOF analysis (Table 3). These analyses aided the identification of a few additional proteins. Some membrane and immunogenic proteins also differed between macrophage- and tick cell-derived bacteria. Immunogenic proteins of vector and nonvector tick cell-derived bacteria had very similar patterns (Table 3). Immunogenic membrane proteins were also evaluated by GeLC-MS/MS. Analysis aided the identification of a few additional proteins, including TFSS protein (VirB9), an ankyrin repeat protein (ECH_0684), and the preprotein translocase (SecA subunit). The VirB9 protein was found only in macrophage-derived E. chaffeensis. Membrane and immunogenic protein analysis led to the identification of only one p28-Omp gene product (p28-Omp14) expressed in tick cell-derived E. chaffeensis; in macrophage-derived bacteria, p28-Omp14, p28-Omp19, and p28-Omp20 were detected. Multiple protein spots were detected for proteins made from p28-Omp14, p28-Omp19, and p28-Omp20 genes and the hypothetical protein gene ECH_0526 (Fig. 3A).


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  		TABLE 3. Immunogenic membrane proteins of E. chaffeensis analyzed by MALDI-TOF

Protein grouping of identified proteins. Shotgun MS analysis of fractionated total proteins and individually picked membrane and immunogenic proteins originating from macrophage and tick cells verified expression of 278 proteins, which represents about one-third of all identified ORFs from the E. chaffeensis genome. Identified proteins were grouped as various functional categories according to the E. chaffeensis annotated genome data (17) (Table 4). Expressed proteins identified by proteomic analysis included proteins from all functional categories except the group of genes listed as disrupted reading frames, which may represent pseudogenes. The majority of expressed proteins were represented in the group of proteins made from hypothetical protein genes, followed by functional groups for protein synthesis and protein fate and energy metabolism. Proteins representing categories of unknown function and cell envelop were also expressed in high numbers.


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  		TABLE 4. Comparison of proteins identified from different functional categoriesa

Subcellular localization of identified proteins. Regardless of whether E. chaffeensis originated from macrophages or tick cells, localization of surveyed proteins by the BII prediction software by Wang et al. (40) positioned about three-fourths of all detected proteins as cytoplasmic proteins (65 to 75%) and one-fourth (22 to 29%) as membrane-associated proteins (Fig. 4). Prediction analysis also placed 3 to 6% of total identified proteins in the group of extracellular (secretory) proteins. A majority of proteins grouped as membrane-associated proteins had molecular masses ranging from 28 to 223 kDa and included proteins previously identified as outer membrane-expressed proteins such as the p28-Omp proteins and 120-kDa glycoprotein. The majority of predicted secretory proteins represented molecular sizes ranging from 20 to 46 kDa but also included some larger proteins of 67, 72, and 303 kDa (Table 5). Predicted secretory proteins included TFSS proteins and several hypothetical proteins. Ankyrin repeat proteins, likely secreted proteins (18, 20), are also expressed by E. chaffeensis.


Figure 4
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  		FIG. 4. Subcellular localization of macrophage (DH82) and ISE6 tick cell culture-derived E. chaffeensis proteins analyzed by the BII web-based program. The number of proteins in each group is given for each portion of the pie chart, and the percentage of the total in each localization group is identified in parentheses.


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  		TABLE 5. Predicted extracellular proteins


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DISCUSSION
 
Shotgun proteomics has been used extensively in recent years to survey the proteins of many organisms (16, 19, 38). For example, this strategy has been valuable in surveying proteomic differences in different life cycle stages of several Plasmodium species (16). In the present study, we identified many expressed proteins of E. chaffeensis by using shotgun proteomics, which involved fractionating purified total proteins of the pathogen originating from macrophages and tick cells and subjecting fractionated proteins to GeLC-MS/MS analysis. We also used GeLC-MS/MS and MALDI-TOF methods to survey individually picked total membrane proteins and immunogenic membrane proteins of E. chaffeensis. We identified a total of 278 E. chaffeensis proteins. The E. chaffeensis genome was sequenced recently (17), and the availability of complete genome sequence data greatly aided the interpretation of our proteomic data. Our study is the first and the most comprehensive proteome analysis of the human monocytic ehrlichiosis pathogen. This also is the first study confirming the expression of nearly one-fourth of all predicted genes of E. chaffeensis, validating that they are functionally active genes, and demonstrates that classic shotgun proteomic approaches are feasible for tick-transmitted intraphagosomal bacteria.

One of the concerns with the proteome analysis, particularly for intracellular bacteria, is host protein contamination. We addressed this concern by using three different approaches. These included the use of a stringent method of purification and performing the protein identification searches using the E. chaffeensis genome data. Although this limits the possibility of identifying any contaminated host proteins, one cannot rule out the false identification of homologous host proteins as the pathogen proteins. To avoid this, we subjected the matched peptides to a database search against the nonredundant database. Our analysis found no evidence for the presence of host proteins in the pool of proteins analyzed. These measures validate that the proteome data reported in the present study represent proteins expressed by E. chaffeensis.

E. chaffeensis resides in tick and vertebrate host cells (11), and the pathogen has two morphologically distinct forms: dense core cells and reticulate cells (4). The dense core form is the infectious form, and the reticulate form is the replicating bacterium that resides in the phagosome of a host cell (31, 42). The pathogen may vary its protein expression to adapt to tick and vertebrate host environments, in support of its intracellular growth and survival, and also to transform from reticulate to dense core, infectious cells. E. chaffeensis protein expression may also be altered in support of evading tick and vertebrate host responses. Intervention strategies will be most effective if targeted to proteins essential for different stages of pathogen growth and those expressed in a host cell-specific manner. E. chaffeensis-expressed proteins in macrophages differed considerably from the pathogen originating from tick cells. Commonly expressed proteins were predominantly made from genes coding for normal physiological functions of a cell (e.g., those involved in protein synthesis, energy metabolism and the biosynthesis of building blocks [amino acids, nucleic acids, and lipids]). Macrophage- and tick cell-specific proteins included many hypothetical proteins, cell envelope proteins, and proteins with unknown function. Proteins identified in the unknown-function group included many novel proteins, such as ankyrin repeat proteins, GTP-binding proteins, zinc finger-like domain proteins, and metallopseudopeptide glycoprotease. The ankyrin repeat protein homologue from A. phagocytophilum has recently been described as a secretory protein (18, 20) and is also considered to play an important role in modulating host response against the pathogen, possibly by interfering with host gene expression (1). GTP-binding proteins and zinc finger proteins play important roles in regulating cell functions and gene expression (6, 7). Expression of these proteins in E. chaffeensis suggests that they may also be important for cellular processes within macrophage and tick cells.

It is possible that some proteins identified as uniquely expressed in the present study may be expressed in both host cell backgrounds. This may be particularly true for a protein that is expressed at a very low level. Although host cell-specific expression of previously detected proteins, such as the p28-Omp proteins (36, 37), validates the relevance of data reported here, host cell-specific expression of a protein of interest must be confirmed by an independent method prior to initiating additional studies. Our study confirmed 55 hypothetical protein genes of E. chaffeensis as truly representing functional genes. Proteins made from hypothetical protein ORFs may represent a unique group of E. chaffeensis proteins that may serve as targets for novel drug and vaccine development.

Previous studies identified few proteins as membrane-associated proteins. In the present study, we identified several more membrane-associated proteins. Our findings also suggest the E. chaffeensis membrane is very complex. Moreover, membrane protein structure appears to differ considerably in bacteria originating from macrophage and tick cells. Immunogenic proteins may also represent an important group of proteins involved in E. chaffeensis interaction with tick and vertebrate hosts (1, 22, 28). Although the proteins identified and listed in Table 3 are likely immunogenic proteins of E. chaffeensis, their immunogenicity needs to be validated independently prior to undertaking detailed immunological studies.

Recent studies suggest that A. phagocytophilum, a rickettsial closely related to E. chaffeensis, uses the TFSS to secrete proteins into host cell cytoplasm (18, 20). However, little is known about the TFSS in E. chaffeensis (27). We identified here 14 putative secretory proteins expressed by E. chaffeensis originating from macrophage and tick cells. In addition, two ankyrin repeat proteins were identified. It will be interesting to determine whether ankyrin repeat proteins are secreted into host cell cytoplasm by the TFSS, similar to the previous reports on A. phagocytophilum (18, 20). Similarly, other predicted secreted proteins may serve as effectors of the TFSS pathway or other, yet-uncharacterized secretory pathways. We presented evidence here for the expression of other secretory pathway proteins, such as the ABC transporter proteins and Sec-dependent and Sec-independent export proteins.

Our previous protein analysis showed that the p28-Omp14 is expressed in tick cell-derived E. chaffeensis, whereas p28-Omp19 and p28-Omp20 are expressed in macrophage-derived E. chaffeensis; we also reported several posttranslationally modified expressed forms of the p28-Omp proteins in E. chaffeensis (36, 37). We made similar observations in the present study when individually picked proteins from 1-DE-resolved membrane and immunogenic proteins were assessed by MS analysis. However, when shotgun proteomic analysis was performed, we identified peptide fragments for 18 of the 22 p28-Omp proteins in macrophage-derived E. chaffeensis; in the tick cell-derived pathogen, expressed proteins from this locus were only from p28-Omp1 and p28-Omp14. The percentage of the identified sequence in the shotgun proteomic analysis was significantly higher for the p28-Omp19 and p28-Omp20 proteins for macrophage-derived E. chaffeensis (see Table S1 in the supplemental material). Similarly, a greater percentage of sequence coverage was observed for the p28-Omp14 protein in the tick cell-derived E. chaffeensis. Our identification of multiple p28-Omp proteins in the present study is similar to recent observations from membrane protein analysis reported by Ge and Rikihisa (14). Previously, Zhang et al. (41) reported the presence of antibodies against all 22 proteins of the p28-Omp locus in dogs infected with E. chaffeensis. Further, RT-PCR analysis of E. chaffeensis RNA isolated from in vitro cultures, in vivo studies in dogs experimentally infected with the pathogen, and A. americanum ticks infected with E. chaffeensis also demonstrated similar differential expression (5, 21, 39). Although the p28-Omp19 in macrophages and p28-Omp14 in tick cells are the major expressed proteins of E. chaffeensis (36, 37), our present findings support previous reports that nearly all 22 p28-Omp proteins are expressed in the vertebrate host environment and only one to two proteins are expressed in the tick cell environment. Although expression appears to be high for one or two proteins made from the p28-Omp locus (p28-Omp19 and p28-Omp20), it is not clear why the bacterium has to express nearly all 22 proteins in vertebrate host cell environment. One possible hypothesis is that expression of the p28-Omp19 is critical for the pathogen's survival in vertebrate host environment and that the expression from other genes at low levels may provide nonessential targets for host immunity, enabling the pathogen to evade host response for its continued survival in a vertebrate host. This hypothesis, however, remains to be tested.

Popov et al. (31) and Zhang et al. (42) demonstrated that the outer membrane 120-kDa glycoprotein is expressed on the infectious dense core form of E. chaffeensis but not in the reticulate form in vertebrate cells. In the present study, we detected this protein only in tick cell-derived E. chaffeensis. We reasoned that the 120-kDa protein expression in macrophage-derived E. chaffeensis is significantly low compared to tick cell-derived organisms. To validate this hypothesis, we performed a semiquantitative RT-PCR on RNA isolated from E. chaffeensis recovered from different time points postinfection from macrophage and tick cell cultures (data not shown). Independent of the time postinfection, the RT-PCR data confirmed high levels of expression from the 120-kDa protein gene in tick cells and also suggested low-level expression in macrophage-derived E. chaffeensis.

Only minor differences were noted in protein expression patterns of E. chaffeensis in vector and nonvector tick cell environments, suggesting that E. chaffeensis protein expression is altered in response to the tick cell environment. Vector and nonvector tick cells are very similar in inducing protein expression differences in E. chaffeensis that are distinct from the macrophage-grown pathogen.

E. chaffeensis is an emerging pathogen, and the functional genomic approach described here aided in the identification of many novel proteins. The availability of protein expression data will be valuable in initiating studies to define the functional significance of the expressed proteins to pathogen infection resulting from a tick bite. Functional genomic approaches, as described here, are crucial for validating genome data (17) and also provide valuable information regarding protein expression. Thus, we provide here a foundation for future studies to map large-scale protein expression differences in the pathogen during growth in the vertebrate and tick hosts, particularly to determine whether similar differences in gene expression will be noted in vivo and also to determine what proteins may be contributing to pathogen evasion mechanisms.


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ACKNOWLEDGMENTS
 
This study was supported by the National Institutes of Health grants AI055052 and AI070908.

We thank the Stanford University Mass Spectrometry Facility, Stanford, CA, for technical assistance for GeLC-MS/MS analysis. We also acknowledge Kamesh Sirigireddy for assisting with Vector NTI software.

This manuscript is Kansas Agricultural Experiment Station contribution 08-224-J.


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

{triangledown} Published ahead of print on 18 August 2008. Back

{dagger} Supplemental material for this article may be found at http://iai.asm.org/. Back

Editor: R. P. Morrison


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Infection and Immunity, November 2008, p. 4823-4832, Vol. 76, No. 11
0019-9567/08/$08.00+0     doi:10.1128/IAI.00484-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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