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Infect Immun. Jul 2010; 78(7): 3047–3052.
Published online May 3, 2010. doi:  10.1128/IAI.00300-10
PMCID: PMC2897376

Identification of Anaplasma marginale Proteins Specifically Upregulated during Colonization of the Tick Vector[down-pointing small open triangle]

Abstract

The transition between infection of the mammalian host and colonization of an arthropod vector is required for the ongoing transmission of a broad array of pathogens, from viruses to protozoa. Understanding how this transition is mediated provides opportunities to disrupt transmission through either chemotherapy or immunization. We used an unbiased proteomic screen to identify Anaplasma marginale proteins specifically upregulated in the tick compared to the mammalian host. Comparative mass spectrometric analysis of proteins separated by two-dimensional gel electrophoresis of uninfected and infected ISE6 cells and infected mammalian cells identified 15 proteins exclusively expressed or upregulated in tick cells. All 15 had originally been annotated as hypothetical proteins. We confirmed quantitative upregulation and expression in situ within the midgut epithelial and salivary gland acinar cells of vector ticks during successful transmission. The results support the hypothesis that A. marginale gene expression is regulated by the specific host environment and, in a broader context, that the core genome evolved in the arthropod vector with differential regulation, allowing adaptation to mammalian hosts. Furthermore, the confirmation of the in situ expression of candidates identified in ISE6 cell lines indicates that this approach may be widely applicable to bacteria in the genera Anaplasma and Ehrlichia, removing a major technical impediment to the identification of new targets for vaccine and chemotherapeutic blocking of transmission.

The transition between infection of the mammalian host and colonization of an arthropod vector is required for ongoing transmission of a broad array of pathogens, from viruses to protozoa. Understanding how this transition is mediated provides opportunities to disrupt transmission through either chemotherapy or immunization. Bacteria in the genera Anaplasma and Ehrlichia are obligate intracellular pathogens and effectively invade, survive, and replicate in markedly different cell types in the mammalian host and ixodid ticks, the arthropod vector (4). Impressively, this transition is effected by using a small genome of <1.5 Mb (2, 3, 8, 9, 15). We and others have hypothesized that the bacterial proteome would be specifically molded for each environment, with a core set of proteins expressed universally and subsets specifically up- or downregulated depending on the host/vector environment (6, 12, 19, 26, 27). However, there has been only minimal proteomic evidence that supports accepting this hypothesis. The best evidence comes from recent analysis of E. chaffeensis that detected proteins present in either in vitro-infected tick cells or canine macrophages (26); however, unique or upregulated expression of these candidate proteins in the tick cells has not been confirmed. There has been no identification of bacterial proteins specifically upregulated or exclusively expressed during actual colonization in the tick.

We addressed this knowledge gap by an unbiased proteomic approach using the St. Maries strain of A. marginale. The St. Maries strain is naturally transmitted by Dermacentor andersoni, in which it colonizes the midgut epithelium after initial acquisition feeding on an infected animal, replicates, invades the salivary gland, and then undergoes a second round of replication during transmission feeding on a new mammalian host (5, 29, 30). Importantly, the complete genome of the St. Maries strain has been sequenced and annotated (2), providing a pathway to identification of expressed proteins using mass spectrometry. The strategy was to first examine the full complement of A. marginale proteins expressed during cultivation in the ISE6 tick cell line. Although this cell line cannot be assumed to represent the actual tick environments of either the midgut or salivary gland, the replication of A. marginale to high titer in ISE6 cells provided sufficient material to conduct a proteome-wide screen to generate a candidate list of proteins (1, 16). The expression levels of these candidate proteins were then compared to in vivo expression levels in the mammalian host and in the tick midgut and salivary gland using both quantitative and in situ localization approaches. We report here the testing of this approach and discuss the findings in the context of the overall hypothesis of proteome regulation at the mammalian host-tick vector interface.

MATERIALS AND METHODS

Proteomic screening for identification of tick stage-specific proteins.

The St. Maries strain of A. marginale, a highly tick-transmissible strain for which the genome has been completely sequenced and annotated (2, 29, 30), was used in all studies. The overall approach to identify candidate A. marginale tick stage-specific proteins was as follows. Bacteria were isolated from infected ISE6 cells, and the bacterial lysate was separated by two-dimensional gel electrophoresis and stained to examine the full complement of proteins. Candidate tick-stage specific bacterial proteins were identified by comparison to proteins separated by two-dimensional electrophoresis of uninfected ISE6 tick cells (to identify and subtract out any contaminating ISE6 cellular proteins) and A. marginale St. Maries strain isolated from infected bovine erythrocytes (to identify and subtract out stage-common bacterial proteins) run under identical conditions.

In detail, A. marginale were isolated by filtration using a 2-μm-pore-size filter (Whatman), as previously described (21), and the washed bacterial pellet was resuspended in phosphate-buffered saline (PBS) containing Complete Mini-Protease inhibitor (Roche). Uninfected ISE6 tick cells were handled identically as a control. Bacteria or uninfected tick cells were lysed in a buffer containing 500 mM Tris, 50 mM EDTA, and 10% NP-40. The lysates were processed with a ReadyPrep 2D cleanup kit (Bio-Rad) and solubilized in 8 M urea, 2% CHAPS {3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate}, 0.2% Bio-Lyte 3/10 ampholytes (Bio-Rad), and 0.001% bromophenol blue. Isoelectric focusing (IEF) was carried out using 11-cm immobilized pH gradient strips under four conditions: a wide-range gradient (pH 3 to 10) and three narrow-range gradients (pH 3 to 6, pH 5 to 8, and pH 7 to 10). Each strip was rehydrated with a total of 150 μg of protein and focused for 35,000 V·h using a Protean IEF cell system. After IEF, second-dimension electrophoresis was performed using 10% polyacrylamide gels. The gels were stained with SYPRO Ruby (Bio-Rad), and individual gel images from infected tick cells, uninfected tick cells, and infected erythrocytes were overlaid to match spots using PD Quest image analysis software (Bio-Rad). Spots identified by either PD Quest or visual inspection as unique to infected tick cells were excised, processed by in-gel trypsin digestion, and identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Confirmation of unique or upregulated tick stage-specific protein expression by quantitative Western blotting.

The three candidate tick-stage-specific proteins with the highest MASCOT (Matrix Science) ion scores after LC-MS/MS analysis—AM410, AM470, and AM829 (Table (Table1)—were1)—were used to confirm differential expression. Each protein was expressed as a His-tagged recombinant protein, affinity purified, and used to immunize mice to generate polyclonal and monoclonal antibodies for use in quantitative Western blot analyses. Briefly, the following primer sets were used in PCR amplification of sequences predicted (http://tools.immuneepitope.org) to encode a B-cell epitope-bearing region of each protein: a 1,035-bp fragment of AM410, 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTAAGCCCATTTAAAAGCAGG-3′ and 5′-GGGGACCACTTTGTACAAGAAAGCTGGGTACTATGCGGACGCTGCGGCCTG-3′; a 1,500-bp fragment of AM470, 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATAGACCCACATTGGCGA-3′ and 5′-GGGGACCACTTTGTACAAGAAAGCTGGGTACTACATCGCCTTCCTTTGCCG-3′; and a 420-bp fragment of AM829, 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTACTGAGCAGAGTGCAGGATATTT-3′ and 5′-GGGGACCACTTTGTACAAGAAAGCTGGGTACTACCGGCGGAACCGTC-3′. The amplicons were cloned and expressed as His-tagged fusion proteins by using a Gateway expression system (Invitrogen). The insert was sequenced using the T7 primer to ensure correct orientation, the correct protein coding sequence, and an in-frame position of the His tag. BL21-A1 Escherichia coli was transformed with the expression plasmid, cultured in LB broth containing 50 μg of carbenicillin/ml, and induced with 0.2% l-arabinose. His-tagged proteins were purified by using a ProBond purification system (Invitrogen).

TABLE 1.
Anaplasma marginale proteins upregulated in tick cell culture

To generate antibodies, mice were immunized and boosted subcutaneously with 50 μg of each recombinant protein emulsified in Titermax Gold adjuvant (CytRx). For monoclonal antibody production, mice were boosted intravenously with 50 μg of antigen without adjuvant 3 days immediately prior to hybridoma fusion. Fusion and limiting dilution cloning were performed as described previously (32). Hybridoma supernatants were screened for reactivity by immunoblotting with A. marginale isolated from infected ISE6 cells. For quantitative Western blotting, A. marginale isolated from each host cell type were quantified by using msp5-based quantitative real-time PCR as previously described (5), and 107 bacteria were loaded per lane. Uninfected ISE6 cells and uninfected erythrocytes were used as negative controls. Electrophoresis was carried out using precast 4 to 20% polyacrylamide gels (Bio-Rad). The proteins were transferred to nitrocellulose membrane and probed with monoclonal antibody AnaF16C1 (reactive with Msp5) as an internal control for equal loading. AM410 and AM470 expression was detected using, respectively, monoclonal antibodies 142/184.8 and 143/694.12.11, while AM829 expression was detected using a 1:500 dilution of specific polyclonal serum. Reactivity was detected by using the Western Star chemiluminescence system (Applied Biosystems). An unrelated isotype-matched monoclonal antibody TRYP1E1 (reactive with a Trypanosoma brucei protein) and a polyclonal serum (1:500 dilution; reactive with a Babesia bovis recombinant protein) were used as negative controls.

In situ expression of unique or upregulated tick-stage specific proteins in Dermacentor andersoni.

In situ expression of AM410, AM470, and AM829 was detected by immunohistochemistry on A. marginale-infected male D. andersoni ticks. An msp5 PCR and Msp5 C-ELISA-seronegative calf (28) was infected by intravenous inoculation of the St. Maries strain. During the acute phase of infection (bacteremia ≥ 108 A. marginale organisms per ml), ticks were acquisition fed for 7 days. Ticks were then removed and incubated at 26°C and 96% relative humidity for 7 days to allow complete digestion of the blood-meal. Ticks were subsequently transmission fed for 7 days on a second naive calf. A cohort of the transmission fed ticks was removed, and the midguts and salivary glands were individually dissected and placed in PBS containing protease inhibitors for Western blot analysis as described above. A second cohort was immediately fixed in 10% formaldehyde and embedded in paraffin. Serial 4-μm sections were deparaffinized, and immunohistochemistry was performed as previously described (29). Serial sections were reacted with 15 μg of each monoclonal antibody/ml or a 1:200 dilution of anti-AM829 polyclonal serum; monoclonal antibody TRYP1E1 or a 1:200 dilution of anti-B. bovis polyclonal serum were used as negative antibody controls. Uninfected ticks, handled identically, were used as a negative antigen control. Binding was detected with horseradish peroxidase-labeled anti-mouse antibody (Dako) and counterstained with Mayer's hematoxylin.

RESULTS

Proteomic screening for identification of tick stage-specific proteins.

As we were seeking to identify A. marginale proteins that were either uniquely expressed or with upregulated expression in tick cells, we used three sets of controls to ensure that the number of organisms isolated from the mammalian host (bovine erythrocytes) was greater than or equal to the number isolated from ISE6 cells. First, we determined the number of organisms isolated from each source by quantitative PCR of msp5, a single-copy gene (2, 5, 31). Second, the quantitative PCR results were confirmed by detection of Msp5, a constitutively expressed protein, in each sample by using Western blotting (Fig. (Fig.1).1). Third, identification of Msp4, an additional constitutively expressed protein encoded by a single-copy gene (2, 23), in the gels following two-dimensional electrophoresis and densitometric quantification using PD Quest image analysis software, revealed no statistically significant difference between host cells (Fig. (Fig.2).2). Msp4 was absent in the uninfected tick cells, as expected (Fig. (Fig.2).2). A total of 16 spots were identified in A. marginale isolated from tick cells and absent in both uninfected tick cells and in A. marginale isolated from bovine erythrocytes (Fig. (Fig.3).3). Of the 16 spots, 10 were identified using the PD Quest software analysis by the overlay of gels and densitometric analysis (unpaired Student t test) revealed statistically significant higher expression (P = 0.01) in the tick cell-derived A. marginale compared to bacteria from infected erythrocytes. The other six spots were identified visually with no detection of a spot in the corresponding gels of A. marginale from infected erythrocytes. Analysis using LC-MS/MS identified 15 unique proteins from the 16 spots. All 15 proteins were mapped to the A. marginale genome; all had previously been annotated as hypothetical proteins (Table (Table1).1). In addition, we detected for the first time the expression of the following proteins as part of the core A. marginale proteome in ISE6 tick cells: AM842 (dnaK), AM944 (groEL), AM254 (tuf), AM666 (atpD), AM956 (pepA), AM880 (alp2), AM564 (mdh), AM937 (fumC), AM326 (argD), AM887 (rpoA), AM735 (infB), AM917 (rpsA), AM418 (pbpA2), AM1313 (virB11), and AM1314 (virB10).

FIG. 1.
Constitutive expression of Msp5 in Anaplasma marginale from infected ISE6 tick cells, bovine erythrocytes, Dermacentor andersoni midgut, and D. andersoni salivary glands. Each lane was loaded with 105.43 ± 0.59 bacteria and reacted with anti-Msp5 ...
FIG. 2.
Identification of Anaplasma marginale proteins uniquely expressed or upregulated in tick cell culture. (a) infected ISE6 cells; (b) uninfected ISE6 cells; (c) infected bovine erythrocytes. Gels were stained with SYPRO Ruby to detect total protein. Circles ...
FIG. 3.
Anaplasma marginale proteins uniquely expressed or upregulated in tick cell culture. Gels were stained with SYPRO Ruby to detect total protein. Circles represent protein spots exclusively present in A. marginale isolated from infected ISE6 cells; the ...

Confirmation of unique or upregulated tick stage-specific protein expression by quantitative Western blotting.

Confirmation of differential expression was examined for the three candidate tick stage-specific proteins with the highest MASCOT ion score following LC-MS/MS analysis: AM410, AM470, and AM829 (Table (Table1).1). Equal numbers (107 ± 0.05) of A. marginale isolated from ISE6 tick cells or from infected erythrocytes were analyzed by immunoblotting with antibodies specific for each candidate protein. Am470 was only detected in the tick cell-derived A. marginale (Fig. (Fig.4).4). Am410 and Am829 were expressed at higher levels in the tick cell-derived A. marginale compared to bacteria isolated from infected erythrocytes (Fig. (Fig.4).4). Densitometric analysis of independent replicates (n = 3) revealed a statistically significant upregulation (unpaired Student t test) for both Am410 (P = 0.0005) and Am829 (P = 0.005) in the tick cell A. marginale. As an internal control, Msp5 levels were similar among all samples (Fig. (Fig.4),4), with no statistically significant difference.

FIG. 4.
Upregulated expression of AM470, AM410, and AM829 in Anaplasma marginale isolated from infected tick cells. A. marginale (107 ± 0.05 organisms) isolated from infected ISE6 tick cells, A. marginale (107 ± 0.05 organisms) isolated from infected ...

In situ expression of unique or upregulated tick-stage specific proteins in Dermacentor andersoni.

To test whether these A. marginale proteins upregulated in the ISE6 cell line were actually expressed in the natural tick vector at the time of transmission, we utilized Western blots using midguts and salivary glands isolated from transmission fed ticks. Am410, Am470, and Am829 expression was detected in 105.6 ± 0.59 A. marginale isolated from infected midguts and salivary glands; there was no detection of these proteins using an equal number of A. marginale from infected erythrocytes or in uninfected erythrocytes and uninfected tick cells (data not shown). To confirm the site of protein expression in situ, immunohistochemistry was performed on the infected, transmission fed ticks. Serial sections of midguts and salivary glands, containing respective means of 105.8 ± 0.59 and 106.1 ± 0.49 A. marginale per organ, revealed the expression of both AM410 and AM470 using monoclonal antibodies and the expression of AM829 using a specific polyclonal antibody (Fig. (Fig.5).5). Serial sections of infected ticks were negative using the unrelated control monoclonal antibody TRYP1E1 or a control polyclonal antibody raised against an unrelated B. bovis protein (Fig. (Fig.5).5). Uninfected ticks were negative in immunohistochemistry with all antibodies (Fig. (Fig.5).5). A. marginale was successfully transmitted by tick feeding with microscopic detection of acute bacteremia 14 days after initiation of tick transmission feeding with confirmation by msp5 PCR (data not shown).

FIG. 5.
Expression of AM470, AM410, and AM829 (arrows) in the midgut (MG) and salivary gland (SG) of Anaplasma marginale-infected Dermacentor andersoni. (a, b, and d) Serial sections of both infected and uninfected ticks probed with monoclonal antibody 143/694.12.11, ...

DISCUSSION

Based on the data, we accept the hypothesis that the A. marginale proteome is specific to the tick vector, with unique and upregulated expression of individual proteins compared to expression in the mammalian host. This in itself is not surprising from either a purely theoretical framework that adaptation to markedly different environments requires a specific proteome or a comparative perspective with other tick-borne bacterial pathogens. Both Borrelia burgdorferi and B. hermsii have been shown to have unique tick-associated gene expression with specific requirements for transmission (7, 24, 25). However, A. marginale differs markedly from Borrelia spp., including the requirement for intracellular replication and the developmental cycle within the tick (10, 11, 29, 30). The identification of specifically upregulated A. marginale proteins in the tick provides candidates for vaccine and drug development and are likely informative for other tick transmitted Anaplasma and Ehrlichia spp.

Technically, the relatively low quantity of bacterial protein within the tick vector has precluded broad proteomic screening. The development of tick cell lines permissive for in vitro growth of Anaplasma and Ehrlichia spp. have removed, in part, this impediment by supporting replication to a high titer and, equally importantly, by allowing incorporation of uninfected cells of the same line as a control (1, 16, 17). The two-dimensional gel electrophoresis approach used in the present study allowed effective discrimination between tick cell and bacterial proteins. The utility of cell lines notwithstanding, how well these cells represent the actual tick cellular environment has been a persistent question. This is illustrated by the use of the ISE6 cell line in the experiments reported here: the cells are derived from embryonic Ixodes scapularis while, in contrast, A. marginale infects, sequentially, midgut epithelial and salivary gland acinar cells in adult ticks of several genera but not including Ixodes (1, 18). The demonstration that A. marginale proteins identified as being upregulated or exclusively expressed in the ISE6 cell line were also expressed in infected D. andersoni indicates that the cell line is a useful predictor of expression in the natural vector, at least to a first-order approximation. This supports the biological relevance of in vitro transcriptome and proteome analysis of other Anaplasma and Ehrlichia spp. (19, 26, 27).

The proteomic approach was unbiased as to the identity, localization within the bacterium, or presumed function of the proteins. We selected this approach for two reasons: (i) there were no comparative data available on tick-borne bacteria in closely related genera that would guide a more targeted approach, and (ii) 30% of the A. marginale genome is annotated as encoding hypothetical proteins (2). That all 15 proteins identified by our approach were originally annotated as hypothetical proteins supports this unbiased methodology. The addition of these 15 proteins to 39 identified in recent studies defining the A. marginale proteome involved in protective immunity extends linkage of the genome annotation to the proteome (14, 21, 22). The progressive confirmation that proteins initially annotated as hypothetical are actually expressed in either the mammalian host or tick vector indicates that these proteins are unique among bacteria with unknown function rather than being erroneous identification of coding sequences. This conclusion is also supported by the linkage of proteome analysis to the genome of E. chaffeensis (9, 26).

A. marginale proteins Am410, Am470, and Am829 were each expressed in both the midgut epithelium and salivary gland acinar cells of transmission fed ticks. Although these three identified proteins segregate by host type, tick versus mammal, we would hypothesize that there are also organ-specific expression phenotypes within the tick. This discrimination, which requires screening of additional tick-specific proteins, may be critically important for discovery of vaccines or drugs that block acquisition (at the level of the midgut) versus transmission (at the level of the salivary gland). None of the three proteins has as yet a demonstrated function in A. marginale. However, an Am410 ortholog has recently been identified in the closely related tick-borne pathogen A. phagocytophilum, APH0859 (originally also annotated as a hypothetical protein, now designated Ats-1). Ats-1 has recently been shown to traffic to the mitochondrion of A. phagocytophilum-infected cells, where it interferes with apoptosis, allowing time for intracellular bacterial replication (20). Unlike A. phagocytophilum, which infects neutrophils in the mammalian host and requires blockage of apoptosis to complete a replicative cycle (20), A. marginale infects non-nucleated mature erythrocytes and thus the need for Ats-1 would be predicted to be dispensable in the bovine host. In contrast, within the tick vector A. marginale must invade and replicate in phagocytic midgut epithelial cells in order to establish colonization (10). Am410 fits this prediction with expression markedly upregulated in the tick vector and expressed in the midgut epithelium. This conservation of gene content between A. marginale and A. phagocytophilum (2, 9), which share common sites of colonization in the tick but differ in the specific hematopoietic lineage infected in the mammalian host (4), is consistent with the theory that bacteria in the family Anaplasmataceae first evolved in arthropod vectors and then diverged as they infected mammals. The differential regulation of this shared gene content, as needed for the specific host environment and cell type, exemplified by Am410 expression, is congruent with but by no means definitive proof of this theory.

All prior data for A. marginale proteins differentially expressed between the mammalian host and tick vector was for downregulated expression (Omp1, Omp4, Omp7 to Omp9, and Omp11; Msp1a) or loss of expression (OpAG3) in tick cells (6, 13, 21, 22). Interestingly, all of these proteins are expressed on the A. marginale surface and exposed to the mammalian immune system. In contrast, only Am778 of the 15 proteins identified in the present study as being exclusively expressed or upregulated in tick cells is predicted to be surface exposed (21). This suggests that interaction with the humoral immune system may be less deterministic in the tick and that evading clearance by innate mechanisms such as phagocytosis and killing or by induced apoptosis may be more important. Both the approach and the newly identified proteins provide opportunities for novel strategies to block tick colonization and subsequent transmission.

Acknowledgments

This study was supported by National Institutes of Health grant AI44005, Wellcome Trust grant GR075800M, and U.S. Department of Agriculture grants ARS 5348-32000-027-00D/-01S and CSREES 35604-15440. S.S.R. was supported primarily by a scholarship from Botswana College of Agriculture, which is an associate institute of the University of Botswana.

Notes

Editor: R. P. Morrison

Footnotes

[down-pointing small open triangle]Published ahead of print on 3 May 2010.

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